CHAPTER 7
PRODUCTS OF MIXED FERMENTATIONS

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Most traditional fermented food products are made by a complex interaction of different micro-organisms. This chapter deals with the products made when there is not a single dominant set of micro-organisms.

7.1 Vinegars

Vinegar is the product of a mixed fermentation of yeast followed by acetic acid bacteria. Vinegar, literally translated as sour wine, is one of the oldest products of fermentation used by man. It is the acetic acid produced by the fermentation of alcohol (ethanol) which gives the characteristic flavour and aroma to vinegar.

It can be made from almost any fermentable carbohydrate source, for example fruits, vegetables, syrups and wine. The basic requirement for vinegar production is a raw material that will undergo an alcoholic fermentation. Apples, pears, grapes, honey, syrups, cereals, hydrolysed starches, beer and wine are all ideal substrates for the production of vinegar. To produce a high quality product it is essential that the raw material is mature, clean and in good condition.

Indigenous vinegars can be made quite simply by the spontaneous fermentation of a fruit or alcohol. All that is necessary is an alcoholic substrate, strains of acetic-acid forming bacteria (acetobacter) and oxygen to enable the oxidation of alcohol. However, this process is very slow and vinegars produced by this method tend to be of inferior quality. Controlled fermentation conditions produce a more acceptable product. A wide range of raw materials can be made into vinegar.

Throughout Asia particularly the Philippines and Sri Lanka

A clear liquid with a distinctive acetic acid taste with a hint of a coconut flavour.

Coconut water is a waste product, which is produced in appreciable quantities in the Philippines, Sri Lanka Thailand and other countries. Its conversion into vinegar therefore presents an attractive option for decreasing wastage and producing a valuable product.

Coconut water is a good base for vinegar, but its sugar content is too low (only about 1%). Sugar needs to be added to bring the level of sugar up to 15%. After the addition of sugar, the coconut juice is allowed to ferment for about seven days, during which time the sugar is converted to alcohol. An alternative method is to pasteurise the coconut water and sugar mixture and add yeast.

After this initial fermentation, strong vinegar (10% v/v) is added to stimulate the growth of acetic acid bacteria and discourage further yeast fermentation. The acetic acid fermentation takes approximately one month, yielding a vinegar with approximately 6% acetic acid. The fermentation will take less time than this if a generator is used.

After fermentation, the vinegar must be stored in anaerobic conditions to prevent spoilage by the oxidation of acetic acid. (Steinkraus, 1996)

Clarification can be achieved by stirring with a well beaten egg white, heating until the egg white coagulates and filtering (Anon).

Latin America and Asia

This product enables the utilisation of pineapple peels, which are usually discarded during the processing or consumption of the fruit. The product has a distinct, very light pineapple flavour and has the same uses as any commercial vinegar.

The peels should be from very well washed ripe pineapples (damaged, rotten or infected fruits should not be used as a source of peels). Use only the peels, not the leaves or stems. The water used should be potable water, boiled if necessary. All the equipment should be well cleaned, as well as the bottles, which should also be steam-sterilised before use.

The peels should be cut into thin strips and put into clay or pewter pots. Aluminium or iron pots should not be used.

Sugar and clean water are added. Each pot is then inoculated and covered with a clean cotton cloth, held around the pot with an adhesive tape, to prevent contamination by insects or dust. The inoculated pineapple is fermented at room temperature (about 20-22⁰C) for about eight days. The acidity should be checked daily. The water level should be maintained during this period. The product should be increasingly acid and by the eighth day it should have the required concentration of 4 per cent acetic acid in vinegar. If higher acidity is desired the product is left to ferment for another one or two days.

The development of acidity should be checked by tasting the product during fermentation.

The residual bacteria removed may be reused as a residue inoculum two or three times more.

The traditional process may be improved by a two-stage fermentation in which alcohol is first formed by yeast (Saccharomyces cerevisiae) and the ‘must’ is then inoculated with acetic acid bacteria (Acetobacter pasteurianus). In outline, the process involves liquidising the peels and diluting with water (water:pulp is 4:1), adjusting the pH to 4.0 using sodium bicarbonate and adding yeast nutrient (ammonium phosphate) at 0.14g per litre. A starter culture is added at 2.7g per litre and the fermentation allowed to take place at 25⁰C for two days. The ‘must’ is then filtered and inoculated with acetic acid bacteria and allowed to ferment for eleven days with aeration of the ‘must’. Other parts of the process are similar. Additional equipment includes a pH meter, refractometer, liquidiser, fermentation locks and equipment for preparing the starter cultures (Fellows, 1997).

Pineapple	Well washed in clean water
Peel the fruit	Take care not to damage hands
Cut the peel	Cut into thin strips and put into clay or pewter pots
Mix with sugar	Sugar is dissolved in clean water
Ferment	Each pot is then inoculated and covered with a clean cotton cloth
Filter	Strain through a cheese cloth
Package

Packaging and storage

The vinegar is bottled in clean glass bottles and stored in a cool dark place.

Palm wine vinegar is a produced across West Africa. It is a vinegar containing about 4% acetic acid, produced from the oxidation of palm wine. It is mainly consumed by people in urban areas as a salad dressing and meat tenderiser, although it also has medicinal uses and is valued in certain rituals. Palm wine is fermented using the same process as for grape wine vinegar – the oxidation of alcohol to acetic acid. The spontaneous process takes about four days. The optimum fermentation temperature is 30º C.

Coconut toddy vinegar is produced throughout South Asia particularly Sri Lanka. It is a clear liquid with a strong acetic acid flavour and a hint of coconut flavour. The fresh toddy is strained, prior to allowing yeast fermentation to occur naturally for 48 to 72 hours. The yeast cells and debris are then removed by progressive sedimentation. After two to four weeks of settling the fermented toddy is placed in barrels. The alcohol is then converted into acetic acid by acetic acid bacteria which are naturally present. The process can be hastened by adding vinegar as a starter. The fermented toddy is converted into vinegar in about three months. Ageing for six months, results in a pleasantly flavoured final product (Jayawardena, 1977).

In East Asia particularly Papua New Guinea a vinegar is made from the sap of the Nipa palm (Nypa fruticans) (Paivoke, Adams and Twiddy, 1984).

Quick process pickles are easy to make but do not really constitute a fermented food product. For this technique, vegetables are soaked in a low salt solution for a few hours. They are then drained and placed in a container. The container is filled with a hot vinegar and spice mixture or a hot oil and spice mixture (Kordylas, 1990). There are hundreds of different recipes utilising locally available fruit and vegetables. For instance the book "Pickles of Bangladesh" has recipes for mango sour pickle, sliced mango pickle, sweet olive pickle, hot olive pickle, sweet tamarind pickle, chalta pickle and green chilli pickle (Azami, 1994).

7.2 Cocoa products

Africa, Asia and Latin America particularly Cote d'Ivoire, Ghana, Indonesia and Brazil.

A fine brown powder with the characteristic taste of cocoa. It is a major ingredient in the confectionery and bakery industries. The product has a short shelf life. "Drinking chocolate" is a mixture of cocoa powder and sugar.

Cocoa beans are the seeds of the cocoa plant (Theobroma cacao). Cocoa pods are cut from the cocoa tree. The pods are cut and the beans removed. Only fully ripe and undamaged beans should be selected. It is important that the beans are processed quickly.

It was formerly believed that cocoa beans were fermented to remove the adhering pulp (Wood, 1990). However a good flavour in the final cocoa or chocolate is dependent on good fermentation. Fermentation is carried out in a variety of ways but all depend on heaping a quantity of fresh beans with their pulp and allowing micro-organisms to produce heat (Beckett, 1988). The majority of beans are fermented in heaps although better results are obtained using boxes, which result in a more even fermentation.

Fermentation lasts from five to six days. During the first day the adhering pulp is liquified and drains away with the temperature rising steadily. The initial alcoholic fermentation gives way to acetification. This and other chemical changes cause the temperature to rise in excess of 50^oC. The beans die. It was thought in the past that death was mainly due to increasing temperature. It is now known that acetic acid at a concentration of 1 percent in the bean is the cause of death and that it is only enhanced by heat, lactic acid and ethanol. The pH value of the cotyledon drops from 6.45 to 4.5 over 120 hours and that during the same period the acetic acid content increased from 0 to 1.36 percent, while the lactic acid content increased from 0.005 to 0.12 percent. When the bean dies maceration of the tissue takes place, allowing enzymes and substrate to mix freely. The possible substrates for enzymes are carbohydrates, lipids, phenolics and amino acids. In addition it is known that the bacteria can metabolise alcohols and organic acids of various kinds. The changing chemical picture is complex. Possible major substrates for micro-organisms are carbohydrates, lipids, phenolics and amino acids. Unlike some flavours and aromas, that of chocolate is not attributable to a single compound (Carr, 1985) (Minifie, 1980).

During fermentation the external appearance of the beans changes. At first they are pinkish with a covering of white mucilage. Gradually the colour darkens and the mucilage disappears. The beans on the surface are always darker than those deeper in the heap or box, indicating that the colour change is oxidative. As the beans are mixed, their colour becomes a more uniform orange-brown and they are only slightly sticky. At this stage they are ready for drying.

The beans need to be dried to a moisture content of less than 7.5%. The beans are dried by either being spread out in the sun in layers a few centimetres thick or in artificial dryers. There are numerous types of dryers but it is important that any smoky products of combustion do not come in contact with the beans otherwise taints will appear in the final product.

The beans are cleaned to remove the extraneous matter.

Cocoa beans consist of an outer skin that needs to be removed and inner "nib". The shell is sometimes removed before roasting and sometimes after roasting.

For cocoa powder roasting temperatures of 120 to 150° C are used. There are many designs of roasters: both batch and continuous systems. The operation is controlled so that the cocoa is heated to the required temperature without burning the shell or the cotyledon. The heat is applied evenly over a long period of up to 90 minutes to produce even roasting. The bean must not be contaminated with any combustion products from the fuel used and provision must be made for the escape of any volatile acids, water vapour and decomposition products of the bean (Wood, 1980) (Cakebread, 1975). After roasting the beans are cooled quickly to prevent scorching.

The roasted nibs are ground into a powder in a plate mill. The resulting powder is sieved through fine silk, nylon or wire mesh.

To produce cocoa powder, some of the cocoa butter needs to be removed. With low fat cocoa powder, more than 90% of the cocoa butter is removed. With medium fat cocoa powder, more than 78% of the cocoa butter has been removed. Finally high fat cocoa powder has less than 78% of the cocoa butter removed. Extrusion, expeller, or screw presses are used in the cocoa industry to remove the cocoa

The cake from the mill is ground in a hammer mill to produce the cocoa powder.

Cocoa beans
Sort	Select only mature beans
Ferment	In heaps or boxes
Clean	Remove extraneous material
Dry	In the sun or in artificial dryers
Roast	120 to 150° C
Grind
Remove cocoa butter
Grind cake
Pack

Packaging and storage

Cocoa powder is hygroscopic (picks up moisture from the air) and should be protected, especially in humid climates. Lidded tins or sealed polythene bags should be used.

Throughout Africa, Asia and Latin America.

A brown solid oily product with the characteristic taste of chocolate. It is a major ingredient in the confectionery and bakery industries.

Cocoa beans are the seeds of the cocoa plant (Theobroma cacao). Cocoa pods are cut from the trees, and the beans are removed from the pods. Only fully ripe and undamaged beans should be selected. It is important that the beans are processed quickly.

Fermentation, drying and cleaning of the beans have been described in Section 7.2.1.
For cocoa butter production the roasting temperatures are 100° C to 104° C There are many designs of roasters: both batch and continuous systems. The operation is controlled so that: the cocoa is heated to the required temperature without burning the shell or the cotyledon The heat is applied evenly over a long period of up to 90 minutes to produce even roasting; the nib must not be contaminated with any combustion products from the fuel used and provision must be made for the escape of any volatile acids, water vapour and decomposition products of the nib (Wood, 1980) After roasting the beans are cooled quickly to prevent scorching

Roasting will have already loosened the shell. The beans are then lightly crushed with the object of preserving large pieces of shell and nib and avoiding the creation of small particles and dust. The cocoa bean without its shell is known as a "cocoa nib". The valuable part of the cocoa bean is the nib, the outer shell being a waste material of little value.

Alkalization is a treatment that is sometimes used before and sometimes after grinding to modify the colour and flavour of the product. This was developed in the Netherlands in the last century and is sometimes known as "Dutching". This involves soaking the nib or the cocoa mass in potassium or sodium carbonate. By varying the ratio of alkali to nib, a wide range of colours of cocoa powder can be produced (Glossop, 1993)^. Complete nib penetration may take an hour. After alkalization the cocoa needs to be dried slowly.

The cocoa nib is ground into "cocoa liquor" (also known as "unsweetened chocolate" or "cocoa mass"). The grinding process generates heat and the dry granular consistency of the nib is turned into a liquid as the high amount of fat contained in the nib melts (Gates, 1990)^..

There are various pre-treatments to develop the flavour of the cocoa mass with and without reaction solutions. These include the "Luwa thin-layer evaporator", "Petzomat thin-layer process", "Cocovap process", "Lehman KFA process" and "Carle-Montanari process" (Beckett, 1988) .

Extrusion, expeller, or screw presses are used in the cocoa industry for the production of cocoa butter from whole beans, and mixtures of fine nib dusts, small nibs, and immature beans. Research in the Kerala Agricultural University has led to develop a suitable pressure device capable of separating cocoa butter from ground cocoa mass ideally suitable for small scale manufacturers (Ganeshan, 1990)^. In Peru a simple screw press is used to extract cocoa butter from beans. The crude cocoa butter is filtered through cloth and allowed to solidify.

To produce plain chocolate, cocoa mass is mixed with sugar and sufficient cocoa butter to enable the chocolate to be moulded. The ratio of mass to sugar varies according to the national taste. The mixture is ground to such a degree that the chocolate is smooth to the palate. At one time this was done by a lengthy process in melengeurs - heavy granite rollers in a revolving granite bed - but nowadays grinding is done in a series of rolls.

The chocolate is then "Conched". This may last for several hours. The chocolate is heated, this helps to drive off volatile acids, thereby reducing acidity when present in the raw bean, and the process finishes the development of flavour and makes the chocolate homogeneous (Wood, 1980) ). Similar processes are involved in the manufacture of milk chocolate. The milk is added in various ways either in powder form to the mixture of mass, sugar and cocoa butter, or by condensing first with sugar, adding the mass and drying this mixture under vacuum. The product is called ‘crumb’ and this is ground and conched in a similar manner to plain chocolate. After conching the chocolate has to be tempered before it is used for moulding or for enrobing confectionery centres. Tempering involves cooling and reaching the right physical state for rapid setting after moulding or enrobing.

7.3 Coffee

Throughout Africa, Asia and Latin America particularly Brazil, Colombia, Indonesia, Mexico and Cote d'Ivoire.

A fine dark brown powder made from roasted coffee beans. Brewed with boiling water and consumed as a drink.

Coffee beans are harvested from two plants Coffea arabica and Coffea canephora variety robusta. Only ripe berries should be used in coffee production. Berries can be placed in water so that immature berries which float can be identified and discarded.

Dry processing is the simpler of the two processing methods and is popular in Brazil for the processing of Robusta coffee and in Sri Lanka for processing Arabica coffee. The coffee cherries are dried immediately after harvest by sun drying on a clean dry floor or on mats. The dried berry is then hulled to remove the pericarp. This can be done by hand using a pestle and mortar or in a mechanical huller. The mechanical hullers usually consist of a steel screw, the pitch of which increases as it approaches the outlet so removing the pericarp. The hulled coffee is cleaned by winnowing.

Wet processing involves squeezing the berry in a pulping machine or pounding in a pestle and mortar to remove the outer fleshy material (mesocarp and exocarp) and leave the bean covered in mucilage. This mucilage is removed by fermentation. Fermentation involves placing the beans in plastic buckets or tanks and allowing them to sit, until the mucilage is broken down. Natural enzymes in the mucilage and yeasts and bacteria in the environment work together to break down the mucilage. The coffee should be stirred occasionally and every so often a handful of beans should be tested by washing in water. If the mucilage can be washed off and the beans feel gritty rather than slippery, the beans are ready.

There is much debate about the fermentation of coffee beans. Some researchers feel that the mucilage breakdown is caused by enzymatic breakdown. For instance Wellman has stated that enymatic fermentation starts immediately the beans have been squeezed from the fresh berries. If these ‘pulped’ beans are piled up or put in a container and protected from any bacterial or other contamination the fermentation will progress. After a number of hours the enzymes of the pulp will have acted on the torn tissues, gorged with starches, sugars and pectins, in such a manner that, without any microbial intervention, the remaining pulp will be easily detached from the beans and washed off in water (Wellman, 1961). However most investigators acknowledge the necessity for the presence of micro-organisms for the depectinisation of the beans.

The following micro-organisms have been isolated: Leuconostoc mesenteroides; Lactobacillus plantarum; Lactobacillus brevis, Streptococcus faecalis, Aerobacter (Enterobacter) and Escherichia, pectinolytic species of Bacillus, Saccharomyces marscianus, S. bayanus and a Flavobacterium sp., Erwinia dissolvens, Fusarium spp, Aspergillus spp and Penicillium. (Pedersen and Breed) (Vaughn et al., 1958) (Hilmer et al.,1965), (Agate and Bhat, 1966)

The beans should then be washed immediately as 'off' flavours develop quickly. To prevent cracking the coffee beans should be dried slowly to 10% moisture content (wet basis). Drying should take place immediately after to prevent 'off' flavours developing. The same drying methods can be used for this as for the dry processed coffee. After drying the coffee should be rested for 8 hours in a well ventilated place. The thin parchment around the coffee is removed either by hand, in a pestle and mortar or in a small huller. The hulled coffee is cleaned by winnowing.

The final flavour of the coffee is heavily dependent on how the beans are roasted. Roasting is a time temperature dependent process. The roasting temperature needs to be about 200° C. The degree of roast is usually assessed visually. One method is to watch the thin white line between the two sides of the bean, when this starts to go brown the coffee is ready. As preferences vary considerably from region to region, a lot of research will need to be done to find the locally acceptable degree of roast. Coffee beans can be roasted in a saucepan as long as they are continually stirred. A small improvement is made by roasting the coffee in sand, as this provides a more even heat. A roaster will produce a higher quality product.

Grinding is a means of adding value to a product. However, it is fraught with difficulties. It is easy to make an assessment of an intact bean, while a ground product presents some difficulty. The fear of adulteration and the use of low quality produce is justified. Because of this there is a great deal of market resistance to ground coffee. This market resistance can only be overcome by consistently producing a good product. There are basically two types of grinders - manual grinders and motorised grinders (anon, 1995).

Coffee cherry
Sort the cherries	Select only mature undamaged beans
Pulp	Skin and pulp removed
Ferment	To remove mucilage
Dry the beans	Sun drying or artificial drying
Roast
Grind
Package

Packaging and storage

Roasted beans can be stored in sacks. Milled beans need to be packaged quickly to prevent the loss of volatile flavour components. The packaging material should be airtight. Polythene is not suitable as it is a low barrier to loss of aroma (Fellows, 1997).

7.4 Other mixed fermentation products

Vanilla is produced in Madagascar, Indonesia and various South Pacific islands. It is a dark brown pod about 20 cm in length. Vanilla is produced by fermenting the pods of the orchids of the genus vanilla. The pods are first sun dried for 24 to 36 hours and then blanched in hot water (65° C) for two to three minutes. The pods are then fermented in boxes and dried again.

7.4.2 Tabasco

Tabasco sauce is made in Mexico and Guatemala. The chilli pods are harvested, ground into a paste and placed in a container with salt. The hot and fiery sauce develops.

In the production of tea, there is a process referred to as fermentation. However microbial activity is not involved in the so-called ‘fermentation’ of tea. The chemical changes are effected by enzymes alone. Fermentation rooms are used where moisture and temperature can be controlled. During fermentation even further darkening of the leaf occurs and the typical aroma develops. By subjective judgement of the aroma’s intensity the period necessary for completion is gauged (Carr, 1985).

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