25 Biotechnology for Production of Fruits, Wines, and Alcohol

J. Maud Kordylas

Fermentation is biotechnology in which desirable microorganisms are used in the production of value-added products of commercial importance. Fermentation occurs in nature in any sugar-containing mash from fruit, berries, honey, or sap tapped from palms. If left exposed in a warm atmosphere, airborne yeasts act on the sugar to convert it into alcohol and carbon dioxide. The making of wines and beers uses this biotechnology under controlled conditions. Alcoholic beverages have been produced for centuries in various societies. They are often central to the most valued personal and social ceremonies of both modern and less literate societies. In such traditional ceremonies as childnaming, marriage feasts, and funerals, alcoholic beverages are often present. In Africa, maize, millet, bananas, honey, palm and bamboo saps, and many fruits are used to ferment nutrient beers and wines. The best known being kaffir beer and palm wines.

Industrial fermentation processes are conducted with selected microorganisms under specified conditions with carefully adjusted nutrient concentrations. The products of fermentation are many: alcohol, glycerol, and carbon dioxide are obtained from yeast fermentation of various sugars. Butyl alcohol, acetone, lactic acid, monosodium glutamate, and acetic acid are products of bacteria action, citric acid, gluconic acid, antibiotics, vitamin B12, and riboflavin are some of the products obtained from mold fermentation.

YEASTS

Yeasts, the main microorganisms involved in alcoholic fermentation, are found throughout the world. More than 8,000 strains of this vegetative microorganism have been classified. About 9 to 10 pure strains, with their subclassifications, are used for the fermentation of grain mashes. These belong to the type Saccharomyces cerevisiae. Each strain has its own characteristics and imparts its special properties to a distillate when used in fermentation. A limited number of yeasts in the classification Saccharomyces ellipsoides are used in the fermentation of wines from which brandy is distilled. The strains used in the fermentation of grain mashes are also used in the fermentation of rum from sugarcane extracts and in beer production. Since yeasts function best in slightly acid medium, the mash, juice, sap, or extract prepared for fermentation must be checked for adequate acidity. If acidity is insufficient, acid or acid-bearing material are added. For distilled liquors, fermentation is carried out at 24° to 29°C for 48 to 96 hours, when the mash or must is ready for distillation. The alcohol content of the fermented must is about 7 to 9 percent.

RAW MATERIALS

Cereals and Starchy Roots

For most distilled liquors, the raw material used is a natural sugar as found in honey, ripe fruit, sugarcane juice, palm sap, beet root, milk, or a substance of amylaceous (starchy) nature that can be easily converted into simple sugars using enzymes present in cereals or through the addition of suitable malted cereal. Maize or corn is the most important grain used as fermentable starchy cereal. Starchy roots and tubers are also used. Industrial production of alcohol from cassava in Brazil has been described by De Menezee (1). The alcohol produced is concentrated in a second distillation column to 97.2 percent and is further dried to 99.9 percent and blended with gasoline for energy purposes.

Malt is important in distilled liquor. In addition to converting starches from other carbohydrates to sugars, malt contains soluble proteins that contribute flavor to the distillate obtained from the fermentation of grain malt mixtures.

Sugarcane

Grown throughout the tropics and semitropics, sugarcane and its products, including cane juices, molasses, and sugar are used to make rum and an alcohol derived from rum. Pressed juice from sugarcane can be used as the base raw material for fermentation, or the juice can be concentrated for sugar production with the molasses residue from sugar crystallization used as a base for alcohol fermentation. Molasses contains about 35 percent sucrose and 15 percent reducing sugars. This gives molasses its principal value as an industrial raw material for fermentation to produce rum. Two or 3 liters of molasses produces I liter of rum. Acetone and butanol also are produced from molasses by fermentation with Clostridium bacteria. Food yeast Torulopsis utitis, is prepared from molasses, as are baker's and brewer's yeasts (2).

Coconut Palm

The coconut palm finds many uses on the tropical islands of the Pacific. Toddy is produced by tapping the unopened flower spathe of the coconut palm. The spathe is bruised slightly by gentle tapping with a small mallet and is tied tightly with fiber to prevent it from opening. It is bent over gradually to allow the toddy to flow into a receptacle. About 5 centimeters is cut from the tip of the spathe after about 3 weeks. Thereafter, a thin slice is shaved off once or twice a day and the exuding sap is collected. Palms are tapped for 8 months of the year and rested for 4 months. The average daily yield per palm is about 2 liters. The yield per spathe varies from 15 to 80 liters, and an average palm can yield 270 liters during 8 months of tapping. The fresh sweet toddy contains 15 to 20 percent total solids, of which 12 to 17.5 percent is sucrose.

Toddy ferments rapidly due to naturally occurring yeasts. Fermented toddy contains about 6 percent alcohol. After 24 hours the toddy contains 4 to 5 percent acetic acid and is unpalatable as a beverage. It can be used for the production of vinegar. Fermented toddy can be distilled to produce arrack. Freshly fermented toddy is used instead of yeast in bread making. Constant tapping of coconut palms for toddy eliminates the nut crop. In 1952 in wine distilleries in Sri Lanka, over 49 million liters of toddy was fermented to give 4.5 million proof liters of arrack (2).

Oil Palm

By tapping the male inflorescence of the oil palm, a sweet sap is obtained. The leaf subtending the immature male inflorescence is removed to provide access, the inflorescence is excised, and thin slices are cut once or twice daily. The exuding sap is funneled into a calabash or a bottle. The fresh sap contains 15 percent sugar. Tapping is done daily for 2 to 3 months, yielding about 3.5 liters of sap per day. The sap ferments by the action of bacteria and natural yeast to produce a beverage with a milky flocculent appearance and a slight sulfurous odor known as palm wine. Palm wine is produced and marketed in considerable quantities in Nigeria.

The sap may be boiled to produce dark-colored sticky sugar or jaggery, which does not keep well. About 9 liters of juice produces I kilogram of jaggery. The fermented sap also yields yeasts and vinegar. A mean annual yield of 4,000 liters of sap per hectare of 150 palms has been recorded in eastern Nigeria. This was estimated to have a value more than double that of oil and kernels from similar palms. Tapping, however, reduces the fruit yield. Sap can also be obtained by tapping the crown of the tree laterally or by felling the palm and drilling a hole through the growing point. Both these methods are very wasteful since they kill the plant. The Palmyra palm yields about 2 liters of palm sap per day. Large palms with several tapped inflorescences give as much as 20 liters per day. A single palm of this type is estimated to produce 12,000 liters of sap during its tapping life.

Fruits

Grapes are the most common fruit used as raw material for alcoholic fermentation. They are used in distilled liquor to make brandy. Historically, wine is the product of fermentation of grape species Vitis vinifera. The high sugar content of most V. vinifera varieties at maturity is the major factor in their selection for use in much of the world's wine production. Their natural sugar content provides the necessary material for fermentation. It is sufficient to produce a wine with an alcohol content of 10 percent or higher. Wines containing less alcohol are unstable because of their sensitivity to bacterial spoilage. The grape's moderate acidity when ripe is also favorable to wine making. The fruit has an acidity of less than 1 percent, calculated as tartaric acid, the main acid in grapes, with a pH of 3.1 to 3.7. The flavor of grapes varies from neutral to strongly aromatic, and the pigment pattern of the skin varies from light greenish-yellow to russet, pink, red, reddish violet, or blue-black. Grapes also contain tannins needed to give bite and taste in the flavor of wines and to protect them from bacteria and possible ill effects if overexposed to the air.

Other fruits can be used to produce wine. When fruits other than grapes are used, the name of the fruit is included, as in papaya or pineapple wine. Apples and citrus fruits with sufficient fermentable sugars are crushed, and the fermentable juices are either pressed out for fermentation or the entire mass is fermented. Tropical fruits such as guava, mangos, pineapple, pawpaw, ripe banana, ripe plantain, tangerine, and cashew fruit also contain fermentable sugars with levels varying from 10 to 20 percent. Overripe plantain pulp was reported to contain 16 to 17 percent fermentable sugar, with the skin containing as much as 30 percent (3).

The tropical climate prevailing in Africa is ideal for the growth and multiplication of microorganisms. The environment is abundant in biomass and in raw materials, which are high in starches and sugars and can be used for fermentation. The available literature is sufficient in information on conditions and control measures required for optimum microbial activity in the various microbial processes. Convincing research results are also available to support utilization of microorganisms in the production of high-quality products of commercial importance. What is lacking, however, is organization of the available information to enable selection of appropriate microbial processes that can be put together to form an integrated system to harness desirable microorganisms as a labor force for industrial exploitation. Below an account is given of an attempt to organize four microbial processes into a production system to produce fruits, wines, and alcohol in an experimental project.

INTEGRATED PRODUCTION SYSTEM

An experimental project was established aimed at providing adequate conditions and control measures in four separate biological subsettings to produce quality products through the action of microorganisms. An attempt was then made to synchronize the activities of the subsettings into an integrated system for the production of fruits, wines, and alcohol with jam production as an integral part of the production system.

The four biotechnological subsettings used were: a compost pile, stimulated microbiological activity in the soil for release of nutrients, yeast activity in extracted fruit juices for the production of wines, and yeast activity in juice extracted from pineapple by-products for the production of alcohol.

Composting

In 1984 a two-compartment wooden structure measuring 2 X 1 x 1 meters was constructed to hold two piles of composting material. Cut grass, straw, dried leaves, and other high-carbon organic wastes were collected from the neighborhood. They were layered with chicken manure to provide a nitrogen source to form compost piles within the compartments. Kitchen waste and, later, wastes from fruit processing were also added to the piles. The piles were kept sufficiently moist by sprinkling with water. To encourage optimum microbiological activity, the piles were aerated by constant turning. Observation of heat generation and the rates at which the piles were digested were used to indicate effective microbial activity. The lack of offensive odor from the piles was considered a sign of adequate control conditions within the piles.

Microbial Activity in Soil

The compost obtained was used to prepare selected sites in a backyard plot measuring 9 x 20 meters that was originally filled with clay soil. The clay soil was removed, and mixed with compost. The mixture was placed into the holes to form raised beds for planting. Two guava seedlings obtained from the research station at Njombe were added to other fruit seedlings nursed in pots. These were transplanted into the prepared sites. As more compost was made available, more fruit seedlings were transplanted into position. By mid 1986 the backyard plot was planted with the following fruit trees: six soursops, five guavas, three pawpaw, eight carambola bushes, one mango, and one avocado pear. The fruit trees were interplanted with plantains, cocoyam, pepper, and a few winged bean plants to form a multistory system as usually obtained in traditional cropping systems in Africa.

Sufficient compost was applied regularly to the soil to encourage microorganisms and other soil dwellers to function and to enhance mycorrhizal fungi association with root hairs, to provide nourishment and protection and for the well-being of the plants. The compost was applied by removing the topsoil around the plant to expose the roots. Two to three loads of compost were distributed evenly around the roots and were covered with the topsoil. Fallen leaves around the yard were raked and used as mulch to cover the top of the disturbed soil to prevent it from eroding away during heavy rains. The leaf mulch was also used to protect the soil surface from the pounding rains. It also kept the soil cool during the dry season and helped to conserve soil moisture when the plants are irrigated. To encourage microbial activity in the soil, no inorganic fertilizer was applied and no pesticides were sprayed anywhere in the yard.

The fertility of the soil around the growing plants was regularly monitored using a two-prong fertilizer analyzer that indicated whether the soil had sufficient nitrogen, potassium, and phosphorus. Where a deficiency was indicated, more compost was applied to the soil. The method of removing the topsoil to apply compost aerated the soil. During the rainy season the edges of the soil around the raised beds were lifted slightly with a fork to allow air in without disturbing the soil. The improvement in soil fertility over the years, the physical appearance of the growing trees, the lack of disease, and later the fruit yield were used as parameters to indicate optimum conditions in the soil that promoted microbial activity. Fruit harvests were recorded daily.

Wine from Fruit Juices

Extracted juices from pawpaw and carambola harvested from the backyard and juice extracted from pineapples obtained from the local market were used to carry out wine-making experiments. The pulp remaining after juice extraction from fruits was used to make jam.

To prevent the growth of undesirable microorganisms, the juice extracts were pasteurized. All utensils, tools, and equipment that came into contact with the wine in making, were sterilized and rinsed thoroughly. No chemicals were used in the preparation of the must. Sufficient amounts of yeast nutrients were added for yeast growth. The pH of the must was adjusted and sufficient sugar was added where needed to produce 11 percent alcohol in the finished wine. A small amount of tannin solution was added to provide bite and flavor to the finished wine. The yeasts used for the first experiments were activated according to the manufacturer's directions. Thereafter, pawpaw, pineapple, and carambola wine yeasts were reserved from wines made. These were kept under refrigeration and used for subsequent wine production. All the wine-making stages - first and second fermentations, raking, storage and aging - were carried out in an air-conditioned room so that constant temperatures could be maintained. Finished wines were bottled, pasteurized, cooled, and corked for storage to age in the bottles.

Alcohol Production from Pineapple

The preparation of pineapples usually produced about 40 to 50 percent waste materials. This was made up of the top crown, the fibrous outside skin, the seeded inner cover, and the hard central core. The crown and the fibrous skin were added to the compost pile. The seeded cover and the central core were crushed and kept frozen until needed for juice extraction for fermentation. The sugar level of the pasteurized juice was checked and sufficient amounts of granulated sugar were added to produce about 12 percent alcohol in the fermented must. The pH of the preparation was also adjusted. The fermented must was then distilled. The temperature of the distillation was carefully controlled so that a high concentration of alcohol could be obtained from one distillation. The bulk of the alcohol collected was over 90 percent concentration. This alcohol was used in experiments with fruits to make aperitif drinks and liquors.

INTEGRATION

The activities of the four microbial processes were synchronized and integrated into an interdependent production system where the subprocesses provided support for each other. The composting setup received wastes from fruit processing. The compost was used to enrich the soil in which the fruit trees were planted. Harvested fruits provided juice extracts for wine making, and by-products from fruit processing provided raw materials for alcohol production. Jams were produced from fruit pulp and were marketed to provide financial support for needed research and to purchase equipment.

RESULTS AND DISCUSSION

Composting

It took about 12 months of composting to arrive at the number of turnings needed, and the correct ratios of high-carbon materials to nitrogenous material required to prepare a compost pile without an ammonia odor. When the correct proportions were used, the compost was completed within 3 weeks during the hot dry weather, and in 4 to 5 weeks during the cool rainy season. Sufficient heat was generated to sterilize the compost, and no odor was detected.

Soil and Fruit Production

It took 2 to 3 years of regular application of compost for the clay in the planted sites to change into dark fluffy soil. Earthworms were seen in the soil after 3 to 4 applications of compost. During the first 3 years the growing plants were constantly affected by plant diseases. The infections diminished, however, as the soil fertility improved. None of the infections were serious enough to require action. The attacks increased during the dry season and again toward the end of the rains, especially during periods when the rains were long and heavy.

Table 1 shows guava, soursop, and carambola yields over the years. After their first bearings, most of the trees lost their seasonality and continued to flower, set fruit, mature, and ripen fruit as long as the weather and soil conditions remained favorable. The rains usually started in March/April and enhanced fruit yield. Thereafter, fruit yields were affected by how heavy the rainy season was and how long it lasted. Flowering and fruit settings were greatly diminished in the guava and the soursop during heavy rains. They were, however, resumed as soon as there was a break in the rains. The next harvests were delayed if the rains were heavy and lasted for a long time. The carambola somehow continued to flower and set fruit during the rainy season as long as there was periodic sunlight.

TABLE 1: Fruit Yields (kilograms), 1986-1991

	Guava				Soursop				Carambola
Year	Jan.-June	July-Dec.	Total	Ave./Tree	Jan.-June	July-Dec.	Total	Ave./Tree	Jan-June	July-Dec.	Total	Ave./Tree
1986	7.2(2)	41.9(2)	49.0(2)	24.50	--	--	--	--	--	--
1987	54.8(2)	76.4(2)	131.2(2)	65.6	163.2(4)	9.9(2)	173.0(4)	43.3	--	0.4(1)	0.4(1)	4.0
1988	86.4(3)	131.1(3)	217.5(3)	72.5	132.4(4)	19.2(4)	151.5(4)	37.9	17.0(6)	46.6(6)	63.6(6)	10.6
1989	109.9(3)	98.5(4)	208.3(4)	52.1	294.1(6)	105.6(5)	394.0(6)	66.2	86.2(8)	135.3(8)	221.5(8)	27.7
1990	165.5(5)	129.5(5)	295.0(5)	59.0	195.7(6)	92.6(5)	286.3(6)	47.7	143.5(8)	135.7(8)	279.0(8)	34.9
1991		116.6(5)			341.2(6)				154.9(8)

( ), number of trees bearing fruit.

Quality was high in guavas and soursop harvested at the beginning of the rains. The fruits were large and well formed and had good flavor. Most of the fruits harvested at the ends of the dry and rainy seasons were smaller, malformed, or diseased. This may be due to the effects of too little or too much water on the health of the plants. Too little water may have affected the activities of microorganisms in the soil, and too much water may have reduced air supply to microorganisms in the soil and leaching of nutrients from the soil. Diminished microbial activity may have affected the well-being of the plants. These assumptions might, however, need to be confirmed through controlled experiments.

The 180-square meter backyard plot yielded sufficient quantities of fruits - guava, soursop, and carambola - to provide raw materials for processing to make jams available on the local market throughout 1989 and thereafter. Carambola yields were also sufficient for wine making. The amount of pawpaw harvested from the backyard was not sufficient, however, for both jam production and wine making. More pawpaw was therefore purchased from the local market to supplement the amount harvested. The quantity of mango obtained from the one mango tree was also not sufficient to keep up with the demand for mango jam on the market. More was obtained from the local market.

Table 2 shows total yields for guava, soursop, carambola, and pawpaw harvested from 1986 to 1990. Although two of the four pawpaw trees died, total yields of fruits from the backyard continued to increase over the years. Yields from crops interplanted among the fruit trees, including pepper, cocoyam, plantain, and winged beans, and from the one avocado tree that started bearing fruit in 1990, when added to those obtained from trees in Table 2, provided an overall yield of over 1 ton from the backyard plot in 1989 and again in 1990.

Wine Production

Wine of acceptable quality were produced from pawpaw, pineapple, and carambola. The wines made were either dry, semidry, or sweet.

TABLE 2 Fruit Yields (kilograms), 1986-1990

Fruit	1986	1987	1988	1989	1990
guava	49.0	131.2	217.5	208.3	295.0
Soursop	-	173.0	151.5	397.0	286.3
Carambola	-	0.4	63.6	221.5	279.0
Pawpaw	-	28.3	100.9	72.6	40.0
Total	49.0	332.9	533.5	899.4	900.3

Although no controlled organoleptic assessment was organized to evaluate the acceptability of the wines, reactions from random individuals who tasted the wines were favorable. Marketing trials will be conducted.

Alcohol Production

Juice extracted from the crushed pineapple core and the inner seeded cover contained sufficient sugar to produce 6.5 to 7 percent alcohol after fermentation. With the addition of extra sugar, however, the alcohol content was increased to 10 percent. A total of 25 liters of over 90 percent concentration alcohol was distilled from 200 liters of discarded wines and 100 liters of fermented pineapple waste extract. Portions of the alcohol were used to carry out experiments to produce aperitif drinks with guava, pineapple, passion fruit, carambola, and ginger. The experiments are still in progress.

BIOTECHNOLOGY PRODUCTION SYSTEM

The integrated bioechnology research and development system is shown in Figure 1. The broken-line arrows indicate units not yet included but for which information has been collected to enable their future integration into the system. The chickens are needed to produce manure for the composting process, with meat and eggs as additional marketable products. Wastewater from fruit processing would be recycled to provide water for irrigation and for composting to economize on the use of potable water for those processes.

From the data collected and from experience gained through the project, the integrated biotechnology production system has many advantages:

· It is environmentally sound: Wastes generated from fruit processing and from the backyard plot are recycled through the composting process to produce organic fertilizer.

· Labor requirements have not been excessive: Once the necessary conditions are met and controls applied for microorganisms to grow and multiply, the productive processes for wine and alcohol production, for composting, and for nutrient release for plant nourishment are carried out with little or no supervision.

· Energy requirements are low: Apart from the energy needed for production of jams and for pasteurization and to run the small-scale equipment used in processing, the integrated production system needs limited amounts of energy input to function. The microbial processes generate their own energy. The need for air conditioning to maintain constant environmental temperatures will likely add to the energy costs.

FIGURE

· The system is sustainable: The interdependency of the microbial subprocesses provides sustainable support to each other with limited input required from outside. Funds generated from the sale of products (jams, wines, apertif drinks) are used to support needed research and to purchase equipment and supplementary produce required to sustain the production of marketable products.

· Only practical research is undertaken: Experiments carried out are those needed to solve immediate problems arising from the production system. These are carried out either to improve the quality of a product, to formulate new products from raw materials or byproducts generated within the system, or to enhance marketability of a product.

· Realistic data is collected for feasibility reports. Production and trial marketing of products from the system have enabled real data to be collected. These are being used to evaluate the system economically and to produce a feasibility report based on actual figures to make decisions on establishing an industry based on the prototype research and development unit.

· Valuable experience has been gained: The project has provided valuable experience in the management of a small enterprise.

CONCLUSIONS AND RECOMMENDATIONS

A good number of efficient microbial processes are available. Sufficient knowledge has been accumulated and information provided on their management and control. If properly selected, synchronized, and integrated, the activities of microorganisms from such processes may be harnessed and used. Their exploitation may be a more promising alternative to large-scale industrial technologies imported from developed countries, which developing countries in Africa cannot afford, sustain, or manage.

The priority for research is, therefore, on selecting the right types of microbial processes that can be put together to form sustainable productive systems, with research trials carried out on prototypes to determine the most economically viable combinations to be adopted for commercial exploitation.

REFERENCES

1. De Menezee, I. J. B. 1978. Alcohol Production from Cassava. Pp. 41-45 in: Cassava Harvesting and Processing. International Development Research Center, Ottawa, Canada.

2. Purseglove, J. W. 1985. Tropical Crops. In: Monocotyledons. England: Longman.

3. Kuboye, A. O., A. B. Oniwinde, and 1. A. Akinrele. 1978. Production of Alcoholic Beverages from Ripe Pineapples, Plantain, and Bananas, Vol. 2, Pp. 78-80. Nigerian Institute of Food Science and Technology. Lagos, Nigeria.