STUDIES ON WINE PRODUCTION FROM COCONUT FRUIT (COCOS NUCIFERA LIN.)
Coconut fruit is a tropical fruit with short shelf-live due to prevailing temperature and humid condition in tropical areas like Benue state Nigeria. Consequently, production of wine from fruit may help reduce the level of postharvest loss and increase the range in type of wines. This study undertook production of wine from coconut fruit. Several parameters were determined. Colony counts of the yeast cells during coconut fermentation in cfu/ml showed the highest colony counts of yeast cells of 4.3 x 103 cfu/ml. the pH of the fermenting wine moved from 4.77 at 0hr to 2.79 at 168hrs suggesting it became more acidic and likely to be more inhibitory to spoilage microorganism and gives fermenting yeast a competitive advantage. The temperature of the fermenting wine did not show major variation from time 0hr to 168hrs, however the specific gravity decreased with increased in time 0hr to 168hrs. The alcoholic content was 5.81 after 168hrs of fermentation period. More research is however required to determine the shelf stability of this wine produced in Nigeria
1.1 Background to the Study
Coconut is the fruit produced by the coconut palm (Cocosnucifera) which belongs to the family of Arecaceae. Coconut is found in tropical regions generally within 22°N and S of the equator and most commonly near the sea coast. The coconut palm is found throughout the west tropical lowlands but will grow anywhere if there are sufficient rain and warmth. It has been known for thousands of years and nobody knows its original home. Perhaps the most likely places are Malaysia or Indonesia, but one thing is certain, it has been spread through the tropics almost entirely by man and has become a typical feature (Stanley, 2012).
There is increasing scientific evidence that supports the role of coconut water in health and medicinal applications. Coconut water is one of the world’s most versatile natural product (Satyabalan, 2012). This beverage is well-known for its health and medicinal value. This contains unique chemical composition of sugars, vitamins, minerals, amino acids and phytohormones and a rich source of electrolytes and natural salts, especially potassium and magnesium. It is low calorie and nearly fat-free, low in sugar as well as containing a little fiber to moderate absorption and is rich in cytokinins, or plant hormones, which have anti-aging, anti-cancer, and anti-thrombolytic effects in humans (Satyabalan, 2011). Through these nutritional values on hand, young coconut water can be considered valuable food product
Plate 1: Coconut blossoms and Coconuts on tree
Among the greatest and genius invention of man is the incessant production of vivacious and edible alcoholic beverages from fermentation of natural fruit juice such as grape in wine -making. Wine has been part of human culture for over 6,000 years, serving dietary and socio-religious functions. Its production takesplace on every continent and has been enjoyed by many people from peasants to kings and its chemicalcomposition is profoundly influenced byenological techniques (Nikhil et al., 2009).It is produced by fermentation of juice of ripe grapes using a microscopic single-celled organism called yeasts (Saccharomyces cerevisae), digest sugars found in fruit juice, producing alcohol and carbon dioxide gas in the process. Other naturally occurring microorganisms may grow in the must or juice, affecting the flavors and aromas of the finished wine. For example, lactic acid bacteria use the acids in wine as a source of energy, reducing the wine’s acidity. These bacteria also produce other aromas and are responsible for the buttery smells that can be found in wine. Sometimes the wine maker restricts the growth of lactic acid bacteria, especially if the wine is already low in acidity or if the buttery character would clash with other aromas of the wine. Acetobacter, another type of bacteria, can spoil the wine by converting ethanol to acetic acid to make vinegar. Wine naturally contains about 79 to 89 percent water, 9 to 21 percent alcohol, less than 1 percent fruit acids, and hundreds of aroma and flavor components in very small amounts. Wines are categorized using a number of different methods. Sometimes they are grouped into different categories by grape variety, region of origin, by color, by the name of the wine maker or viticulturist, or by production technique (Bissonet al., 2009).There are two types of wines namelyNatural wines with 9-14% alcohol and Fortified (Dessert and appetizer) wines with 15 to 21% alcohol (Nduka, 2007). The nutritional role of wine is important since its average contribution to total energy intake is estimated to be 10 to20% in adult males (Macraeet al., 1993). Although grapes are the most common fruit used to make winefor the past few decades. In lieu of this several studies have investigated the suitability of other fruits as substrates for the purpose of wine production (Okunowoet al., 2005). Moreover, the non-availability of grapes, which is usually the fruit of choice for wine production in the tropics has necessitated the search for alternative fruit source in tropical countries; Nigeria (Alobo and Offonry, 2009). Wine is also made from the fermented juice of pears, apples, berries, blackcurrants and even flowers such as dandelions are sometimes used.However, are usually referred to as fruits or country wine (Bissonet al., 2009). Any fruit with good proportion of sugar may be used in producing wine and the resultant wine is normally named after the fruit. The type of wine to be produced dictates the fruit and strain of yeast to be involved (Amerine and Kunkee, 2005). In contrast to most foods and beverages that spoil quickly or that can spread diseases, wine does not spoil if stored properly. This implies that the production of wine from fruits is a form of extending the shelf life of the fruit hence, preservation.
Wine composition and quality are functions of many different intrinsic and extrinsic variables, many of which are microbiologically mediated. A large diversity of microbes are inherent to winemaking including various yeasts, bacteria and fungi. Prominent in this process are Saccharomyces species (predominantlySaccharomyces cerevisiae), which dominate the alcoholic fermentation, and the lactic acid bacteria (LAB), which carry out the malolactic conversion. Efforts to determine the population size and potential impact of different microbes on the winemaking process are critical to production of a flavourful product.
Spoilage is considered growth of organisms that are unwanted at any particular place and time in the winemaking process (Sponholz, 1993). Thus the same microorganism can be both beneficial and detrimental to the winemaking process. For example, growth of S. cerevisiae is required during the alcoholic fermentation, but growth can be detrimental if it occurs in a finished, and bottled, wine. Interestingly, both academics and winemakers have good reason to be interested in the microbial ecology of the winemaking process. From an academic perspective wine represents an ideal landscape in which to study basic concepts of microbial ecology. Several factors promote this view. First and foremost, wine is a liquid medium that allows samples to be properly mixed prior to analysis, these contrasts with the situation for those studying microbial growth on solid surfaces (e.g., barrel stave, grape surface or vineyard soil) in which the microbial populations are heterogeneous and spatially distributed across the surfaces. As a consequence, representative sampling of microbes on surfaces becomes a more statistically challenging process than sampling of a liquid medium like must or wine. A second reason why wine is an attractive platform for microbial ecology is the diversity of microbes present which enables one to witness a range of microbial interactions from commensalisms and neutralisms to antagonisms. From the winemaker perspective, close monitoring of the microbial changes occurring throughout the winemaking process is beneficial for several reasons: to promote and guide yeast during the alcoholic fermentationand ultimately to ensure the stability of the wine before bottling and storage (Delfini and Formica 2001).
The evolution of undesired microbes during different stages of winemaking can produce volatile acidity, off-flavors and polysaccharide hazes, all of which can diminish the quality and acceptability of the final product (Sponholz, 1993).
Prior to the onset of fermentation, the Coconuts themselves can be infected with moulds, yeasts and bacteria that can enter and alter the fermentation in a negative fashion. Improper wine storageand handling post-fermentation can encourage microbiological faults, which can negatively impact wine quality. As a result the winemaker must conduct basic physical, chemical, sensory and microbiological analyses of musts and wines to assure wine quality. Whether it is for an investigation of basic ecological concepts or for the applied goal of predicting possible wine spoilage, one must have accurate and reproducible methods for enumeration of various microbial constituents at different stages in wine production. Both indirect and direct approaches can be used to view these populations.
Fermentation of food for preservation, enhancement ofnutritive values, improvement of flavor and preparation ofbeverages has been practiced probably since prehistoric times by people of nearly every civilization (Sofos, 1993).The word fermentation comes from the Latin verb fevere, which means to boil. Which describes the appearance of the action of yeast on extracts of fruit or malted grain during the production of alcoholic beveragesthis appearance is due to production of carbon dioxide bubble caused by the anaerobic catabolism of sugar present in the extracts (Stanbury et al., 2003)during the process complex organic substances are broken down into simpler form? However, fermentation is interpreted differently by microbiologists and biochemists. Toa biochemist the word means process relating to the generation of energy by the catabolism of organic compound, whereas it meaning to a microbiologist tend to be much broader. Which include the microbial physiology view, the industrial principle and it application in food processing; the microbial physiology involve the metabolism of organic carbon to generate energy by substrate level phosphorylation and in which organic molecule generated during catabolism of this carbon compound serve as the electron acceptor (reducing agent).while in industrial principle the culturing of microorganism on a large scale, irrespective of the reducing agent this may involve the aerobic phase of production i.e. industrial production of antibiotics and powdered (dried) yeast cell, also those that involve the anaerobic phase of production include alcoholic beverages production and ethanol production both are physiologically refers to as fermentation. The fermentation of food substances e.g. milk, bread etc. by microorganism such as lactic acid bacteria, has play an important role in the preservation of the fermented foods and as well as increased the flavor, nutrients, digestibility, security and other characteristics of the food(Barnum, 1998).
Fermentation is a process of extracting energy from the oxidation of organic compounds such as carbohydrates using an endogenous electron acceptor, usually pyruvate, an organic compound. Before fermentation takes place, one glucose molecule is broken down into two pyruvate molecules during glycolysis. Fermentation is important in anaerobic conditions when there is no oxidative phosphorylation to maintain the production of Adenosine tri-phosphate (ATP) by glycolysis. Duringalcoholic fermentation, usually carried out by yeasts, pyruvate is then converted into ethanol and carbon dioxide thus:
C6H12O6 → 2C2H5OH+2CO2
During this process, the carboxylic carbon atom is released in the form of carbon-dioxide with the remaining components becoming acetaldehyde. The acetaldehyde in the absence of oxygen will then be further reduced by alcohol dehydrogenase to form ethanol along with carbon-dioxide (Robinson, 2006).It is a way for a cell to obtain energy without using oxygen.
Tropical citrus fruits have been used assubstrates for the production of wines and thealcohol profiles. Variousfactors influence the fermentation process anddetermine the end products obtained. Theseinclude substrate related factors such ascultivar types, cultivation conditions, conditionsat harvest and post harvest handling.
Yeast species are used in many industrialfermentation processes including alcoholicbeverages production. The quality of wineproduced greatly depends on the yeast strain; development of improved starterorganisms for fermentation of citrus juice may offer a relative simple avenue for reducingpost harvest wastage of citrus fruits in lowutilization environment and in places where theproduction of citrus concentrates is low ornonexistent. Juice concentrates are readilystorable and can be used for productionprocesses even when the fruit is out of season. In an effort aimed at increasingthe low industrial utilization and reducing thehigh wastage of Coconut (Cocos nucifera) fruitsin the developing world, and investigated thepossibility of exploiting the fermentative abilityof yeasts to produce Coconut wines. The quantity of alcohol present in Coconutwines produced from musts fermented bySaccharomyces cerevisae weredetermined.
Citrus is the most economicallyimportant tree fruit crop in the world. The majorityof citrus arrives to market in the form ofprocessed products, such as single strengthCoconut juice and frozen juice concentrates. InFlorida, one of the world’s top producing regions,more than 96% of all Coconuts are processed into range juice. Inthe1999–2000 season, Floridaproduced more than four billion litres of singlestrength Coconut juice and as such, the Floridacitrus juice industry is considered an importantfood processing industry in the United States.Economic losses due to juice spoilage areminimized by good sanitation procedures beforeand during citrus processing. Pasteurization,concentration, or low temperature storageprotocols help to reduce the number ofmicroorganisms in the final product. However,these products are not free of microbiologicalspoilage problems, especially non pasteurizedsingle-strength juices. Citrus juices are acidicbeverages (pH3to4) with high Sugar content(15°Brix). Under these conditions, acidolacticbacteria, molds, and yeasts comprise the typicalmicrobiota Present In citrus juices. Potentiality of Rhizopus Oryzae to utilize Coconutpeels under solid state fermentation conditions toproduce macerating fluid with high cellulolytic andpectinolytic activities.
Tropical fruits have been used as substrates for the production of wines (Maldonado et al., 1975; Anuna et al., 1990; Ndip et al., 2001; Osho, 2005; Okunowo et al., 2005). Various factors influence the fermentation process and determine the end products obtained. These include substrate related factors such as cultivar types, cultivation conditions, conditions at harvest and post harvest handling (Daudt and Ough, 1973; Bell et al., 1979; Liu, 2002; Jonathan and Errol, 2000; Joshi and Sandhu, 2000; Kourkoutas et al., 2005). Though the fermentation of fruit sugar usually yields ethanol as the predominant alcohol, small quantities of other higher alcohols (referred to as fusel oil) are also produced from the oxidative deamination, decarboxylation and reduction of amino acids and sugar degradation (Anuna and Akpapunam, 1995). The presence of pectin in some fruits may also result in methanol generation in the fermenting wort (Anuna and Akpapunam, 1995). The alcohol profile is a significant factor in the quality of wines. (Drawert and Rapp, 1966; Anuna and Akpapunam, 1995).
Yeast species are used in many industrial fermentation processes including alcoholic beverages production. The quality of wine produced greatly depends on the yeast strain (Kunkee, 1984; Okunowo et al., 2005). Development of improved starter organisms for fermentation of citrus juice may offer a relative simple avenue for reducing post harvest wastage of citrus fruits in low utilization environment and in places where the production of citrus concentrates is low or nonexistent. Juice concentrates are readily storable and can be used for production processes even when the fruit is out of season (Ramachandra and Arun, 2005; Siddik et al., 2006). For example, in India alone an estimated loss of around 35 000 million Indian Rupees (around 638 million US $) worth fruits and vegetables was recorded despite the fact that India produces around 60 to 65 million tons of fruits and vegetables (Ramachandra and Arun, 2005). While reports indicate that about fifty percent of citrus fruit goes to waste in Nigeria (Jennifer, 1999). In an effort aimed at increasing the low industrial utilization and reducing the high wastage of Coconut (Cocos nucifera) fruits in the developing world, investigationshows the possibility of exploiting the fermentative ability of yeasts to produce Coconut wines (Okunowo et al., 2005).
Chilakaet al., (2010) evaluated the efficiency of yeast isolates from palm wine in diverse fruit wine production. Passion fruit, watermelon and Pineapple were the fruits used for the evaluation. The research conclude that the test fruits were good substrates for wine production and the palm wine yeast isolates did not differ significantly in alcohol production from the commercial yeast.Nwachukwuet al., (2006) investigate on the characterization of yeast isolated from pal wine for industrial utilization for production of wine base on certain attributes necessary for ethanol production, the attributes investigate includes ethanol tolerance and sedimentation rate nine strains of S.cerevisiae, two strains of S.globosus were isolated. The alcohol in wine, ethanol is present in sufficient concentration to kill pathogenic microorganism, which makes it to be considered to be safer to drink than water or milk (Bisson and Butzkc, 2007). In spite of several prohibitional laws on alcohol consumption in some human communities, a number and varieties of alcoholic beverages have been developed, refined and extolled in extensive and rich literatures (Macraeet al., 1993).
Palm wine is the fermented sap of the tropical plants ofthe palmaefamily. It is produced and consumed in verylarge quantities in the south-eastern Nigeria. It containsnutritionally important components including amino acids,proteins, vitamins and sugars (Okafor, 1987). Palm wine is a traditional wine extracted from palm trees Elaeisguineenis and Raphiahookeri.Although palm wine may be presented in a variety of flavours, rangingfrom sweet (unfermented) to sour (fermented) and vinegary, it is mostly enjoyed by people when sweet (Elijah et al., 2010). Palm wine is consumed for its nutritional effect because of its probiotic content (Ezeronye, 2004; Lourens-Hattingh and Wiljoen, 2001; Heller, 2001). Palm wine is characterized by an effervescence of gas resulting from the fermentation of the sucrose content (Bassir, 1962), by the fermenting organisms. These organisms have also been reported to originate from several sources, which include tapping equipment, containers, the environment, etc. (Faparunsi and Bassir, 1972a). Palm wine is consumed throughout the tropics and appears as a whitish liquid. The unfermented sap is clean, sweet, colourless syrup containing about 10 – 12% sugar, which is mainly sucrose (Bassir, 1962; Okafor, 1975a). The abundant nutritional component of palm wine makes this wine a veritable medium for the growth of a consortium of microorganism, whose growth in turn, change the physicochemical condition of the wine. Upon fermentation by the natural microbial flora, the sugar level decreases rapidly as it is converted to alcohol and other products (Obire, 2005). Meanwhile, the sap becomes milky-white due to the increased microbial suspension resulting from the prolific growth of the fermenting organism (Okafor, 1975a,b). Palm wine is produced and consumed in very large quantities in the south eastern Nigeria. Many workers have indeed carried out studies aimed at isolating and exploiting palm wine yeasts for industrial processes. These include for baking, portable ethanol production and single cell protein production. Ogbonna (1984) and Onyedinma (1983) used palm wine isolates of Saccharomyces cerevisiaeto produce artificial palm wine and beer, respectively.The fermentation of wine is known to be a complex process with various ecological and biochemical processes involving yeast strain (Fleet, 2003). Most studies on palm wine have reported its potentials as source of yeast isolate for the fermentation industries.
As modern fungal taxonomy would have it, classic ethanolic fermentations are carried out by strains of the yeast, Saccharomyces cerevisiae, regardless of whether the end product is ale, lager, wine, or a distilled beverage. Over the years, there has been a variety of specific synonyms given to certain industrial strains of this fungus that have been used to produce specific alcoholic beverages, and some of these names have elevated the organism concerned to species status. Examples are Saccharomyces carlsbergensis/Saccharomyces uvarum for the bottom-fermenting yeast used in lager beer production, and Saccharomyces ellipsoideus for the wine yeast. Such names have now largely disappeared from the literature, and are only used by microbiologists in the specialized fields in which they have been relevant historically. In their monumental study, Barnett et al., (2000)report 678 recognized yeast species for which cultures and the results of standard physiological tests were available. The same work provides a register of almost 4000 taxonomic names that have been used at various times, since Hansen’s work, together with their provenances and synonyms. The register is important, because some yeast names have changed so regularly that it is difficult for non-systematic to know which species is being referred to in different publications. Like other plants, the naming of yeasts is nowgoverned by the International Code for Botanical Nomenclature, and the accepted name for the major wine yeast is: S. Cerevisiaevar. ellipsoideus. Barnettet al.,(2000).Use the following scheme for classifying this organism:
SPECIES: S. cerevisiae
In a novel approach to the taxonomy of S. cerevisiae, NaumovProposed six cultivars:
(i) ‘cerevisiae’, for brewer’s top-fermenting yeasts, and baker’sand distiller’s strains;
(ii) ‘ellipsoideus’, for strains from primary wine fermentations;
(iii) ‘cheresanus’, for strains from secondary wine fermentations
(E.g. those forming a film on sherry during oxidation ofethanol);
(iv) ‘oviformis’, for wine yeasts that do not ferment galactose,and that are resistant to high concentrations of ethanol andsulfites;
(v) ‘diastaticus’, for strains able to metabolisedextrins (e.g.soluble starch);
(vi) ‘Logos’, for strains able to ferment melibiose.
Saccharomyces cerevisiae is a eukaryotic microorganism. It appears microscopically as a globuse cell. The yeast forms one to four ascospores, which are smooth and ellipsoidal. Colonies appear smooth, raised and translucent (Martini and Martini, 1998). Saccharomyces cerevisiae can ferment glucose and sucrose. Saccharomyces cerevisiae cannot utilize pentoses (Kudoet al., 1988). On a dry weight basis, Saccharomyces cerevisiae contains 3% to 5% phosphate, 2.5% potassium, 0.3% to 0.4% magnesium, 0.5% sulphur and trace amounts of calcium, chlorine, copper, iron, zinc and manganese (Monk, 1994; Walker, 1998). The organism must be supplied with a source of phosphate, which is incorporated into nucleic acids, phospholipids, adenosine triphosphate (ATP) and other compounds. Potassium is necessary for the uptake of phosphate, and a deficiency may be linked to sluggish alcoholic fermentations (Kudoet al., 1988). Other minerals needed by the organism during fermentation have a variety of functions but are used primarily as enzyme activators. The yeast has been found on the surfaces of plants, the gastro-intestinal tracts and body surfaces of insects and warm-blooded animals, soils from all regions of the world and even in aquatic environments (Martini, 1993). Most often it is found in areas where fermentation can occur, such as on the surface of fruits, storage cellars and on the equipment used during fermentation process (Mortimer, 2000). S. cerevisiae is famously known for its role in food production. It is the critical component of fermentation process that converts sugar into alcohol; an ingredient shared in wine, beer and distilled beverages. S. cerevisiae can exist in two different forms: haploid and diploid. It is usually found in the diploid form (Laundry et al., 2006). The diploid form is ellipsoid-shaped with a diameter of 5-6 micron meters, while the haploid form is more spherical with a diameter of 4 micron meters. Both forms reproduce asexually in a process called budding. S. cerevisiaecan multiplyeither asexually by vegetative multiplication orsexually by forming ascospores.As a eukaryote, S. cerevisiae contains membrane-bound organelles. Its chromosomes are located in the nucleus, and it uses mitochondria to conduct cellular respiration. Like all other fungi, the cell’s shape is based on its cell wall. The cell wall protects the cell from its environment as well as from any changes in osmotic pressure. The inner cell wall has a high concentration of β-glucans, while the outer cell wall has a high concentration of mannoprotein. Chitin is usually located in the septum (Cabibet al., 1991). S.cerevisiae can live in both aerobic as well as anaerobic conditions. In the presence of oxygen, yeast can undergo aerobic respiration, where glucose is broken to CO2 and ATP is produced by protons falling down their gradient to an ATPase. When oxygen is lacking, yeast only get their energy from glycolysis and the sugar is instead converted into ethanol, a less efficient process than aerobic respiration. This aspect of their respiration is the attribute of S. cerevisiae that is being taken advantage of in the industrial world. The main source of carbon and energy is glucose, and when glucose concentrations are high enough, gene expression of enzymes used in respiration are repressed and fermentation takes over respiration. This series of reactions, transforming glucose into pyruvate with the formation of ATP, constitutes a quasi-universal pathway in biological systems. The elucidation of the different steps of glycolysis is intimately associated with the birth of modernbiochemistry. Saccharomyces cerevisiae contains all the essential amino acids, 14 minerals, and 17 vitamins. It is one of the best natural sources of the B-complex vitamins thiamin, riboflavin, niacin, B6, pantothenic acid, biotin, and folic acid. It is also high in minerals, including chromium, zinc, iron, phosphorus, and selenium. Saccharomyces is also a good source of protein. It contains approximately 16 g of protein per 30 g of powdered yeast. Brewer’s yeast is a good source of RNA, an immune-enhancing nucleic acid that may help in the prevention of degenerative diseases and slowing the aging process (Griffith, 2000).
AIM OF THE STUDY
To establishthe possibility of producing suitable wine from Coconut fruits using saccharomyces cerevisiae and to monitor the physicochemical changes occurring during the fermentation