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galactaric acid to inhibit fermentation (yeast)



galactaric acid to inhibit fermentation (yeast)
CHAPTER ONE
1.0    INTRODUCTION.
Fermentation is a process which takes palace due to some certain factors which can also be controlled by some other biochemical reactions. The useful physiological properties of yeast have led to their use in the field of biotechnology; fermentation of sugars by yeast is the oldest and largest application of this technology. The significant source of inhibiting fermentations in other to extend or elongate the shelf life of some industrial and domestic produce continues to be important to people that has more interest in improving the development of our industries and economic values. Though many industries rely on this same fermentation to carry out their daily productions such as the beer producing company, win producing company, bakeries, yogurt producing industries and so many more.
In this work the process of inhibiting fermentation is carried out using galactaric acid known as mucic acid which can also be called meso-galactaric acid obtained by nitric acid oxidation of galactose or galactose- containing compounds like lactose, dulcite, quercite, and most varieties of gum. It forms a crystalline powder which melts at 213 °C. It is insoluble in alcohol, and nearly insoluble in cold water.  This a process in which glucose comes in play with yeast in other for normal yeast action on sugar to occur which is fermentation and the galactaric acid plays it own role when introduced in to the mixture of  yeast and glucose. This work is carried out by observing their sugar level.
1.1   AIMS AND OBJECTIVES
Using galactaric acid to inhibit fermentation (yeast).














CHATER TWO
  2.0 DESCRIPTION OF YAEST
Yeast are eukaryotic micro-organisms classified in the kingdom fungi with the 1500 species currently described, estimated to be only 1% of all yeast species most reproduce asexually by budding, although a fewer do so by binary fission. Yeast is unicellular, though some species with yeast form may become multicellular through the formation of a string of connected budding known as pseudohyphae or false hyphae. Yeast form a single taxonomic or phylogenetic yeast grouping. The term yeast “is often taken as synonym for saccharomyces ccerevisiac but the phylogenetic diversity of yeast is shown by their placement in two separate phyla, the ascomycota and basidomycota (Dujon, 1996).
2.1    SCIENTICCLASSIFICATION OF YEAST.
1. Acomycota
        I.            Saccharomycotina (true yeast)
     II.            Taphinomycotina
   III.            Schizosaccharomycetes (fission yeast)
2.  Basidomycotina
I.            Tremellomycet
II.            Agaricomycotina
III.            Pucciniomycotina
IV.            Microbotryomycetes
                                                                                                       
2.2     HISTORY OF YAEST
The world yeast comes to us from the old English gist and form the indo-European root yes-meaning boil, foam, or bubble. Yeast microbes are probably one of the earliest domesticated organisms. People have used yeast for fermentation and baking throughout history. Archaeologists digging Egyptian ruins found early grinding stones and baking chambers for yeasted bread, as well as drawing of 4, 000 years-old bakeries and breweries, in 1680 the Dutch naturalist Anton Van Leeuwenhoek first microscopically observed yeast. But at that time did not consider them to be living organism, but rather globular structure (Ashwell, 2003). In 1857 French microbiologist lavis Pasteur proved in the paper memoire sur la fermentation alcoolique that alcoholic, fermentation was conducted by living yeast and not by a chemical catalyst (Pasteur showed that by bubbling oxygen in to the yeast broth, cell growth could be increased. But the fermentation in inhibited an observation later called the Pasteur Effect. But the late 1700s, two yeast strains used in brewing had been identified, saccharomyces cerevisiae, so called “high “or top yeast and S.carisbergensis low or bottom yeast. High yeast was sold commercially by the Dutch for bread making starting in 1780, while around 1800; the Germans started producing S.cerevisiae in the form of cream. In 1825 a method was developed to remove the liquid so the yeast could be prepared as solid blocks (Barnett, 2003). In the industrial production of yeast blocks was enhanced by introduction of the filter press in 1867. In 1872, Baron Maxde Springer developed a manufacturing process to create granulated yeast, a technique that was used until the First World War. in the united state, naturally occurring airborne yeast were used almost exclusively until commercial yeast was marketed at the centennial explosion in 1876 in Philadelphia, where Charles L Fleischmann exhibited the product and a process to use it, as well as serving the resultant baked bread (Cumming, et al., 2001).
.
2.3     GROWTH
Yeast is chemo-organotrophs as they use organic compound as a source of energy and do not require sunlight to grow. Carbon is obtained mostly from hexose sugar such as glucose and fructose, or disaccharide such as sucrose and maltose. Some species can metabolize pentose sugar like ribose, alcohols, and organic acids (Nelson, et al., 2000). Yeast species are either requires oxygen for aerobic cellular respiration (obligate aerobes), or an anaerobic but also have anaerobic methods of energy production (facultative anaerobes). Unlike bacteria, there are known yeast species that grow only anaerobically (obligate anaerobes). Yeast grows best in a natural or slightly acidic PH environment. Yeast are generally grown in the laboratory on solid growth media or in liquid broths common media used for the cultivation of yeast include potato dextrose agar (PDA) or potato dextrose broth, Wallenstein laboratories nutrient (WLN) agar, yeast peptone dextrose agar and yeast mould agar or broth (YM). A home brewer who cultivates yeast frequently use dried malt extract and agar as a solid growth medium. The antibiotic cyclo heximide is sometimes added to yeast growth media to inhibit the growth of saccharomyces yeasts and select for wild/indigenous yeast species. This will change the yeast process.

2.4        ECOLOGY
Yeast are very common in the environment, but are usually isolated from sugar-rich materials. Examples includes natural occurring yeast on the skin of fruits and berries (such as grapes, apples or peaches), and exudates from plant such as plant saps or cacti). Some yeast is found in association with soil and insects. The ecological function and biodiversity of yeast are relatively unknown compared to these of other micro-organism (Rastall, et al., 1998). Yeast including Candida albicans, Rhodotorula rubra, Torulopsis, and Trichosporon cutaneum has been found in-between peoples toes as part of their skin flora. Yeast is also present in the gut flora of mammals and some insect. And even deep-sea environment host as array of yeast. Black yeast has been recorded as a partner complex relationship between ants, their mutualastic fungus, a fungal parasite of fungus and bacterium that kills the parasite. The yeast has negative effect on the bacteria that normally produce antibiotic to kill the parasite and so many affect the ant’s health by allowing the parasite to spread (Nelson, et al., 2000).
2.5       REPRODUCTUION
Yeast has sexual asexual reproductive cycles. The most common mode of vegetative growth in yeast is asexual reproduction by budding. Here a small bud or daughter cell is formed on the parent cell. The nucleus of the parent cell splits in to a daughter nucleus and migrates in to the daughter cell. The bud continues grow until it separate from the parent cell, forming a new cell. Some yeast, including schizosaccharomyces pombe, reproduces by binary fission instead of budding. Application of this technology, many types of yeast is used for making many foods (. Baker’s yeast in bread production, yeast in win fermentation and for xylitol production (Dujon, 1996). So called red rice yeast in actually a mold, monascus purpureus. Yeast includes some of the most widely used model organism for genetic and cell biology.

2.6       USES OF YEAST
2.6.1   ALCOHOLIC BEVERAGE
Alcoholic beverages are defined as beverages that contain ethanol (C2H5OH). This ethanol is almost always produced by fermentation, the metabolism of carbohydrate by certain species of yeast under anaerobic or low oxygen condition. Beverages such as wine, beer, or distilled spirits all use yeast at some stage of their production (Cumming, et al., 2001).

A.      BEER
Brewer’s yeast (also known as brewing yeast) can mean any live yeast used in brewing, dried and killed, and used as a dietary supplement for its B vitamin content. Brewers classify yeast as top fermenting and bottom fermenting. This distinction was introduced by the Dane Emil Christian Hansen. “Top fermenting yeasts” are so called because they form foam at the top of the wort during fermentation (Reed, et al., 1991). They can produce higher alcohol concentration and prefer higher temperatures. Producing fruiter, sweeter, ale-type beers, an example of top fermenting yeast is saccharomyces cerevisiae, known to brewers as ale-yeast. Bottom fermenting yeast are typically used to produce larger type of beers, though they can also produce ale-type beers. These yeast ferment more sugars leaving crisper taste, and grow well at low temperature. An example of bottom yeast is saccharomyces pastorianus formerly known as saccharomyces carlsbergenesis. For both types, yeast is fully distrusted through the beer w from hile is fermenting, and both equally flocculate (clump together and participate to the bottom of the vessel) when fermentation is finished. By knows means do all top fermenting yeast demonstrate this behavior, but it features strongly in many English ale-yeast which may also inhibit chain forming (the failure of budded cell to break from the mother cell) which technically different from true flocculation.
In industrial brewing, to ensure purity of strain, a clean sample of the yeast is stored in the laboratory. After a certain number of fermentation cycle, full scale propagation is produced from this laboratory. Typically, it’s grown up in about three or four stages using sterile brewing wort and oxygen. The common top fermenting brewer yeast saccharomyces cerevisiae is the same species as the common baking yeast. However, baking and brewing yeast typically belong different strains cultivate to favor different characteristics, baking yeast strain are more aggressive , in order to carbonate dough in the shortest amount of time possible, brewing yeast strain acts slower but tends to produce fewer off flavor and tolerate higher alcohol concentration (with some strain, up to 22%) (Ashwell, 2003).

2.6.2       DESTILLED ALCOHOL
Distillation: is a method of separating of mixtures based on differences in their volatilities in a boiling liquid mixture. Distillation is a unit operation, or a physical separation process, and not a chemical reaction. Commercially, distillation has a number of applications. It is used to separate crude oil into more fractions for specific uses such as transport, power generation and heating. Water is distilled to remove impurities, such as salt from seawater. Air is distilled to separate its components—notably oxygen, nitrogen and argon for industrial use. Distillation of fermented solution has been used since ancient times to produce distilled beverages with higher alcohol content. The premises where distillation is carried out, especially distillation of alcohol are known as a distillery (Rastall, et al., 1998).
A distilled beverage is a beverage that contains ethanol that has been purified by distillation. Carbohydrate-containing plant material is fermented by yeast, producing a dilute solution ethanol in the process (Cumming, et al., 2001). Spirits such as whisky and rum are prepared by distilling those dilute solutions of ethanol. Components other than ethanol are collected in the condenselate, including water, esters, and other alcohols which accounts for the flavor of the beverage.
A. WINE
Yeast is used in wine making where it controls sugars present in grape juice or must in to alcohol. Yeast is normally already invisible present on the grape. The fermentation can be done with this endogenous wide yeast. However, this may give unpredictable result depending on the exact type of yeast species present, for this reason a pure yeast culture is generally added to the must, which rapidly comes to demonstrate fermentation. This represses wild yeast and ensures a reliable and predictable fermentation (Ashwell, 2003).

2.7         GENERAL DESCRIPTION & APPLICATION OF GALACTARIC ACID
Mucic aid is a trivial name of galactaric acid. If both terminal group of an aldose are oxidized to carboxylic acids, the product is called an aldaric acid. Aldonic is a carboxylic acid resulting from oxidation of only one aldehyde group in aldose ((Bananas, et al., 2003). The suffix aric refers to dicarboxylic sugar acid, (where –onic is for a carboxylic sugar acid, such as ribonic acid derived from D-ribose). Galactaric acid is the carboxylic sugar acid resulting from oxidation of galactose with dilute nitric acid. Glucaric acid is a aldaric acid resulting from the oxidation of glucose. Due to the disclose of latent symmetry in the remaining molecule, they are achiral. Mucic acid is s white crystalline powder melting at 2300C, practically insoluble in (cold) water and alcohol. Chemically it is 2, 3, 4, 5-tetrahydroxyhexanedioic acid. It’s converted to furoic acid, pyranone and thiophene molecules in nature. Produces its anti oxidative and heavy metal chelation activities are applicable in cosmetics and skin care products for anti aging, acne treatment and sun light protection like alpha-hydroxy acid (Cumming, et al., 2001).
.   
2.8      CHEMISTRY OF GALACTARIC ACID
Mucic acid, C6H10O8 or HOOC-(CHOH)4-COOH, (also known as galactaric or meso-galactaric acid) is obtained by nitric acid oxidation of galactose or galactose-containing compounds like lactose, dulcite, quercite, and most varieties of gum. It forms a crystalline powder which melts at 213 °C ((Bananas, et al., 2003.).  It is insoluble in alcohol, and nearly insoluble in cold water. Due to the symmetry in the molecule, it is optically inactive even though it has chiral carbon atoms (i.e., it). When heated with pyridine to 140 °C, it is converted into allommic acid. When digested with fuming hydrochloric acid for some time it is converted into a furfural dicarboxylic acid while on heating with barium sulfide it is transformed into athiophene carboxylic acid. The ammonium salt yields on dry distillation carbon dioxide, ammonia, pyrrol and other substances. The acid when fused with caustic alkalis yields oxalic acid. With potassium bisulfate mucic acid forms 3-hydroxy-2-pyrone by dehydration and decarboxylation’

The interaction of Cu2+ with galactaric acid leads to the formation, at acidic pH, of the complex species (Gala=galactaric dianion), while at physiological pH the prevailing species is Cu(GalaH−2) where the carbohydrate acts as chelating agent toward the metal ion through the carboxylic groups and the deprotonated α-hydroxylic oxygen’s (Brink, et al., 1994). The crystal structures of galactaric acid. And of the ternary complex -bipyridine are also reported. A comparison of the coordinative behavior of galactaric acid and other aldonic and alduronic acids.




2.8.1       STRUCTURE OF GALACTARIC

                     


The crystal structure has a system of strong, intermolecular hydrogen-bonds, which accounts for the high crystal density and low solubility in water.

2.8.2       MOLECULAR FORMULA
Galactaric acid, C6H10O8

 2.8.3      IUPAC NAME
2, 3, 4, 5-tetrahydroxyhexanedioic acid

2.8.4     DERIVATIVES
Aldaric acid is a group of sugar acids characterized by the formula HOOC-(CHOH)n-COOH. Aldaric acids are usually synthesized by the oxidation of aldoses with nitric acid. In this reaction it is the open-chain (polyhydroxyaldehyde) form of the sugar that reacts .An aldaric acid is an aldose in which both the hydorxly function of the terminal carbon and the aldehyde function of the first carbon have been fully oxidized to carboxylic acid functions (Kurtzman, 2006). (Oxidation of just the aldehyde yields an aldonic acid while oxidation of just the terminal hydroxyl group yields an uronic acid.) Aldaric acids cannot form cyclic hemicacetla like unoxidized sugars, but they can sometimes form lactose. Nomenclature of the aldaric acids is based on the sugars from which they are derived; for example, glucose is oxidized to glucaric acid and xylose to xylaric acid. Unlike their parent sugars, aldaric acids have the same functional group at both ends of their carbon chain; therefore, two different sugars can yield the same aldaric acid (this can be understood by looking at the Fischer projection of a sugar upside down—with normal aldoses, this is a different compound due to the aldehyde function at the top and the hydroxyl function at the bottom, but with aldaric acids, there is a carboxylic acid function on both ends, so upside down and right side up do not matter). For example, D-glucaric acid and L-gularic acid are the same compound. A consequence of this is that some aldaric acids are meso forms with no optical activity despite their multiple chiral centers—this occurs if a sugar and its enantiomer oxidize to the same aldaric acid. An example is D-galactose--it has four chiral centers, but D-galactaric and L-galactaric acids (see mucic acid), which have the opposite configuration at each chiral center and therefore would be expected to be enantiomer, are actually the same compound; therefore, galactaric acid is an achiral meso form with no optical activity. Again, this can be understood by taking the Fischer projection of either acid and looking at it upside down—the configuration is now switched at every carbon (Eleguezobal, 2005).

STRUCTURE OF DERIVATIVES
Aldipic acid, HOOC-(CH2)4-COOH is not an aldaric acid, though it is structurally similar. In fact, six-carbon aldaric acids can be considered tetra hydroxyl derivatives of adipic acid.
OXIDATION TO ALDONIC, DIACRBOXYLIC AND URONIC ACID
Under milled conditions, e.g. with bromine water in buffer neutral or alkaline media. Aldoses are oxidized to aldonic acids. Oxidation involves lactol group exclusively. Bpyranose is oxidized more rapidly than the a-form. Since the B-form is more acidic it can be considered that the pyranose anion is the reactive form. The oxidation product is the omega lactone which is in equilibrium with the gamma lactone and the free form of aldonic acid. The transition of lactones from omega to gamma forms and vice versa and probably proceeds through an intermediary bicyclic form. The acid name is obtained by the suffix-onic acid (e.g. aldose→ aldonic acid). Glucono-Ω-lactone is utilized in food when a slow acid release is required, as in baking powder, raw fermented sausages or diary product (Bananas, et al 2003.).
The treatment of aldose more vigorous oxidizing agent, such as nitric acid, bring about oxidation of the C-L aldehyde group and the CH2OH-group resulting in formation of dicarboxylic acid (nomenclature: stem name of the parent sugar + the suffix-aric acid, (e.g. aldose→ aldonic acid). Thus, galactaric acid (common or trivial name: mucic acid) is obtained from galactose.
The carboxylic acid can, depending on its configuration, form mono or dilactones. Oxidations of the CH2OH-group by retaining the carboxyl function at C-L, with the aim obtaining uronic acids (aldehydecarboxylic acids). It’s possible only by protecting the carboxyl group during oxidation. A suitable way is to temporarily block the vieinal HO-group by ketal formation which, after oxidation at C-6 carbon is completed, are deblocked under mild conditions. An additional possibility for uronic acid synthesis is the reduction of monlactones of the corresponding aldaric acid (Brink, et al., 1994).            


2.9         SYNTHESIS
The synthesis of galactaric acid acetate bis alkylation (thiocarbonyl), hydrazide is described. Selective cyclisation of both hydrazides was investigated. Phosphorous oxychloride as cyclising agent led to dehydrative cyclisation and produced 1, 2, 3, 4-tetra-O-acetyl-1, 4-bis (5-S-methyl or — benzyl) 1, 3, 4-thiadiazol-2-yl galactotetritol (Brink. et al., 1994). While thionyl chloride led to dehydrosulfurization and gave 1, 2, 3, 4-tetra-O-acetyl-1, 4-bis (5-S-methyl or -benzyl)-1, 3, 4-oxadiazol-2-yl galactotetritol. Finally with triethyl orthoformate as cyclising agent, compounds, gave 3, 3′-(2, 3, 4, 5-tetra-O-acetyl-galactar-1.6 dioyl) bis (2-ethoxy-2, 3-dihydro-5-S-methyl or benzyl 1, 3, 4-thiadiazole).
2.10       METABOLISM
D-Galacturonic acid-1-C14 is metabolized by the detached ripening strawberry fruit to -ascorbic acid-6-C14, -galactonic acid-6-C14, galactaric acid containing C14 in the carboxyl carbon, and -xylosc-1-C14. In addition, it is incorporated into pectin as anhydro units of -galacturonic acid-1-C14. The methyl ester of -galacturonic acid-U-C14 is utilized by the strawberry for -ascorbic acid synthesis and pectin formation. The relative metabolic distribution of C14 from the free acid and its methyl ester are compared (Prentice, 1991).
Galactaric acid is one of the polyacids playing an important role in processes that occur in human and animal bodies. It interaction with metal ions and the structure of metal galactarate, have been by x-ray diffraction and IR spectroscopy. Complex formation of galactaric with metal ion in solution has been studied insufficiently, probably because this acid is poorly soluble in water and other polar solvents (Eleguezobal, 2005).  

2.11     INERACTION WITH COPPER
The interaction of Cu2+ with galactaric acid leads to the formation, at acidic pH, of the complex species [CuGala] (Gala=galactaric dianion), while at physiological pH the prevailing species is [Cu(GalaH−2)]2− where the carbohydrate acts as chelating agent toward the metal ion through the carboxylic groups and the deprotonated α-hydroxylic oxygen’s (Prentice, 1991). The crystal structures of galactaric acid and of the ternary complex are also reported. A comparison of the coordinative behavior of galactaric acid and other aldonic and alduronic acids is also discussed.
A solution study on the ability of galactaric acid HOOC(CH)(4)COOH] in the complexation of biological metal ions such as Co(II) and Ni(II) and toxic metal ions such as Cd(II), Pb(II) and Hg(II), is reported. The stability constants of the complex species are determined by means of potentiometric measurements. Galactaric acid behaves as chelate ligand through carboxylic oxygen and alpha-hydroxyl group towards Co (II) and Ni(II), while in the Pb(II) and Cd(II) containing system it co-ordinates the metal ion with carboxylic oxygen and two alcoholic hydroxyl groups. The prevailing species at acidic or neutral pH is [MGala] which is also isolated in the solid state and characterized by means of IR spectroscopy. On increasing pH, the [MGalaH(-1)](-) species is also formed where the co-ordinated OH group undergoes deprotonation in all metal ion complexes except those with Hg(II), where the co-ordination of hydroxide ion is suggested as the precipitation of the metal hydroxide occurs at pH 7 (Cumming,  et al., 2001).
2.12      CONDENSATION OF GALACTARIC ACID
Condensation of galactaric acid bis’ hydrazide with carbon disulfide in the presence of ethanolic potassium hydroxide gave the dipotassium salt of galactaric acid bis (hydrazidocarbodithioic acid. Heterocyclization of the key compound 2 produced three different types of double headed a cyclo C-nucleosides: acid-catalyzed dehydrative cyclization afforded the 5-thioxo-1, 3, 4-thiadiazoline 3, base-catalyzed dehydrosulfurative cyclization gave the 5-thioxo-1,3,4-oxadiazoline and condensative cyclization with concomitant dehydrosulfuration and dehydration with different nitrogen nucleophiles yielded the 5-thioxo-1,2,4-triazolines 7. Acetylation of the prepared acyclo C-nucleosides 3, 5, 7 and with acetic anhydride in the presence of pyridine at ambient temperature caused acetylation of the sugar hydroxyls as well as heterocyclo imino protons to give the tetra-O-acetates. Representative members of the prepared compounds were tested for antimicrobial activity (Brink. et al., 1994.).
Keywords: 1, 3, 4-Oxadiazoline; 1, 3, 4-thiadiazoline; 1, 2, 4-triazoline; acyclo C-nucleosides; antimicrobial activity; cyclization; dipotassium salt of galactaric acid bis (hydrazidocarbodithioic acid.Ascomycetous yeasts are maintained on glucose-peptone-yeast            extract agar   (GPYA).
Many basidiomycetous types of yeast do not survive well on the glucose-peptone medium, although they grow well on it. Such yeasts are kept on potato-dextrose agar (PDA). Most strains are stored at temperatures between 4 and 12°C and subcultured at intervals of c. 6 months. Some yeast, for instance Arxiozyma and Malassezia, have to be subcultured every month. Dekker a and Brettanomyces produce excessive amounts of acetic acid, therefore 2% of calcium carbonate is added to the medium to neutralize the acid. Nevertheless, these yeasts still need to be subcultured every two months.
Cultures are frozen in either liquid nitrogen or a mechanical freezer at temperatures between -80° and -135°C for long-term storage. Freezing gives good results at the Centraalbureau voor Schimmelcultures. Cultures of all strains held are frozen and are successfully kept at -80°C and in liquid nitrogen. The preparation of cultures for freezing is simple and quick. The general method used is as follows: short lengths of polypropylene drinking straws are sealed at one end, labeled with a black felt-tipped pen (e.g. Pentel Permanent Marker), and sterilized in the autoclave at 121°C for 15 min. The strain to be frozen is grown for about 24 hr in 3.0 ml of liquid medium on a shaker before adding 1.0 ml of a 60 % solution of glycerol in water. An amount of the resulting suspension is pipetted into the straws sufficient to half fill them. The straws are then closed by clamping the open ends in the jaws of a sealing machine for plastic packages. The cultures are then either frozen at approximately -30°C for between 30 and 60 min before being placed in the storage tank under liquid nitrogen, or put directly into a freezer cabinet at -80°C. Sterile plastic ampoules suitable for use in liquid nitrogen can be bought but are more expensive and take up more storage space
(Prentice, 1991).
CHAPTER THREE
 3.0       SOURCE OF RAW MATERILAS
 The raw materials such as chemicals and reagents used in this very project research work were bought at science and laboratory shops at ogbette main market in Enugu and were of analytical grade, and identified by Mr. Moses Ezenwali (HOD Biochemistry Department Caritas University Enugu) while others were obtained from Biochemistry laboratory faculty of natural science caritas University Enugu.









                                              
3.1                                                Washing Sample Bottle

Weighing Of Reagents



Preparation of Galactaric Acid


                Weighing                                   Preparation Of Glucose Solution
                 Reagents                                      With yeast
                    
                    

                 Heating
                                                                                                          Weighing
                                        
                    Drying                                                                           Dissolving

                                                                                                              Mixture
 

                    Galactaric acid                                          
                                                                                                             Glucose + yeast solution

FLOW CHART FOR SAMPLE PREPARATION

3.2       EQUIPMENT USED
Ø  Electronic weighing balance (g)
Ø  Refractomater
Ø  Burning cylinder
Ø  Conical flask (ml)
Ø  Test tube
Ø  Beaker
Ø  Measuring cylinder (ml)
Ø  Syringe (ml)
Ø  bottles
METHODS
Some steps were carried out before caring out the major practical, they as follow
The bottle was soaked in water containing enough detergents, and were washed properly with washing brush and rinsed with distilled water in the laboratory and allowed to dry properly for couple of two days including the cover to avoid any alteration.
3.3             PREPARATION OF GALACTARIC ACID.
PRINCIPLE
The acid (Nitric acid) is been added to water not water to the acid in other to avoid explosion of acid.  
PROCEDURE
Using electronic weighing balance, 40g of galactose was weighed, with 40ml of concentrated nitric acid using measuring cylinder and 400ml of water were also measured with measuring cylinder for accurate measurement. After the proper weighing of the above listed reagents the mixing process kicks off, by introducing the already weighed acid in the volume of weighed water, then the quantity of the weighed galactose are introduced in to the mixture giving a solution of  (water + nitric acid and galactose). The solution is now transferred in to a beaker for heating.  
The heating cylinders were used to heat the prepared mixtures. In this process the mixture were placed on wire gauze on top of the heating cylinder in a prescribed temperature for a specific period of time. This very mixture continues to heat until the nitric acid present in the mixture evaporates properly. After which it was assumed that the galactaric acid in question has been prepared.
3.4        PREPARATION OF GLUCOSE SOLUTION WITH BAKERS YEAST
PRINCIIPLE.
The sugar level of the glucose were first measured using refractometer, before adding the baker yeast in to the glucose solution in other to note the sugar level of the glucose solution before yeast starts acting on the glucose solution which results to fermentation.

PROCEDURE
600g of baker’s yeast were weighed with electronic weighing balance, after which 200g of glucose were also measured using the same electronic weighing balance; the baker yeast and glucose were dissolved with 2500 ml of distilled water in a clean laboratory container. Before then, the sugar level of glucose before adding the baker yeast were checked with refractometer and noted down.
FIG .1 FOR CONVERSION UINTS OF GALACTARIC ACID IN GRAMS (g)
S/N
SAMPLES
CONCETRATION OF GALACTARIC ACID
1
A
0.05g
2
B
0.1g
3
C
0.2g
4
D
0.3g
5
E
0.4g
6
F
0.5g
7
G
0.6g
8
H
0.7g
9
I
0.8g
10
J
0.9g
11
K
1.0g
12
L
1.5g
13
M
2.0g
Since the unit of measurement of the electronic balance used are in gram (g) some mathematical calculation were carried out during the weighing of the sample (galactaric acid) which is tabulated in the tale above.
After transferring the known concentration of galactaric acid as listed above in to the various labeled samples, the mixture of  glucose and baker yeast solution prepared were induced in to the samples containing different concentration of galactaric acid. And shake as fast as possible to avoid settling, the samples were kept and covered in good laboratory condition after which reading commence the next day.

3.5     ANALYSIS FOR WEIGHT OF CARBON-DIOXIDE (CO2)
Checking the weight of co2 present in each sample is a very delicate and careful step.
PROCEDURE
Each sample was weighed using electronic weighing balance. The initial weight of the sample was noted down, after which the sample container (bottle) cover were removed and the final weight were also taken down. So in other to check for the weight of co2 present, the initial weights were subtracted from the final weight.

MATHEMATICAL EXPRESION
Initial weight = y
Final weight = x
Weight of co2 = k
.:y (initial) – x (final) = k (weight of co2).

 3.5.1      ANALYSIS FOR VOLUME OF CARBON-DIOXIDE
After checking for the weight of carbon-dioxide (co2) then it calls for calculating the volume of carbon-dioxide (co2) in each samples in relationship with the weight of the samples over the constant density of carbon dioxide
Mathematically:
Density = weight ÷ volume
Therefore volume = weight ÷ density
Constant density of carbon-dioxide = 1.98g/ml



3.5.2     ANALYSIS FOR SUGAR LEVEL OF SAMPLES
After calculating the volumes of the carbon-dioxide present then, the reading for glucose level takes place.
PROCEDURE
In checking glucose level, few drop of the sample were collected and dropped on the sensitive lens of the Refractomater, facing the Refractomater towards the reflection light  the sugar level of the sample will be viewed and recorded then the refractometer lens were cleaned with cotton wool before dropping that of another sample in other to avoid error. It continues until the sugar level for all the samples were noted down for six (6) days.








                                             CHARPTERFOUR
4.0      RESULTS
The values in the graphs present in this chapter are expressed as standard results of this laboratory research work.
Fiq.1

GRAHPICAL RESULTS 
Fig 1.
 Fig1. implies that thire is an effect of galactraric acid in the sugar level in sample H.
According to this graphical expression there is an decrease in sugar level of this very sample but to a certain level with respect to the days it becomes constant as shown in the graph below.
Fig 2




The graphical representations of this very sample (J) show know difference with that of sample I which implies that galactaric acid has played a role.
Fig 3




This result of sample k complies with the above shown result of other samples, as well to say, fermentation is being inhibited with respect to the days for fermentation.
Fig 4.



 Fig 5 show a little difference compared to the above displayed graphical results of other samples.
Fig 5


Sample M which is the last result still show that at certain point with respect to days of fermentation, the sugar level was constant.  
Fig6

CHAPTER FIVE
5.0      DISCUSSION OF RESULTS
For decades fermentation has been a process which occurs in everyday life like industries such as breweries, bakeries, wine producing industries and many others. As much as fermentation is required in many industries, it inhibition is also essential in so many other ways in other to prolong the shelf life of products.
Yeast playing an important role in fermentation makes it centre of focus when considering inhibition of fermentation due to the major energy production in yeast is glucose and glycolysis is the general pathway for conversion of glucose to pyruvate. The pyruvate can distinguish from respiration and fermentation, so if all this process which leads to fermentation can be bridged that means fermentation has been inhibited.
Having a result of this research were all the analyzed samples maintains constant result in the level of sugar during fermentation with respect to the applied sample (galactaric acid) thus, there was decrease in sugar level showing sugar break down by yeast (fermentation) but at a certain stage it maintained a constant result of  sugar level.        


5.1      CONCLUSION
Conclusively, the graphical result of this research work shows that there was an decrease in sugar level with respect to fermentation but to a certain stage with respect to the number of days. 
 Backing up this graphical result shown in the above chapter in relationship with the values in the tables which shows the decrease in values of sugar level and constant values maintained in sugar level by almost all the samples implies that there was effect of galactaric acid in the samples which was able to with hold the fast fermenting process of yeast.
 Therefore galactaric acid was able to inhibit the fast process in fermentation which is normally carried out by yeast in the absence of galactaric acid.

RECOMMENDATION
From the experiment carried out so far galactaric acid was able to play an important role by inhibiting the fast action of on yeast on sugar in other for fermentation process to be slowed.
This very aspect of research work is advisable for more research work to be carried out in a professional level with respect to proper development in the area of biochemistry.    

REFERENCES

Ashwell, M. (2003). Concepts of functional food. Nature, 218:415 -417.

Brink, H.N., Buts, K.B. and Raedemarks, M.C. (1994). Saccharomyces Boulardi,
          Enhances Intestinal Enzyme Expression. Pediatrics, 36:522-527.

Barnett, J.A. (2003). Beginning of Microbiology and Biochemistry. Microbiology,
         149:557 – 567.
            
Bananas, P.M., Bass, M.A. and Brown, N. (2003). Yeast form Dominates Fungal
                 Diversity. Retrieved 14 July, 2010 from http://www.google.com

Cummings, J.H., Mc Farland, G.T. and Englyst, H.N. (2001). Prebiotics Digestion and          
                  Fermentation. Clin. Nutrit. 73: 415 – 420

Dujon, B. (1996). The Yeast Genome Project. J. Basic Microbiology, 43: 430 – 436.

 Eleguezobal, J.R. (2005). Molecular Genetics of Yeast. A Practical
             Approach to Yeast. Cambridge: Harvard University Press.

FAO. (2001). Health and Nutritional in Food. Retrieved 6 July, 2010 from                      
        http://www.google.com

Guthric, G. I. and Fink, K. S. (1991). Yeast Species.  Guide to Yeast
          Genetic and Molecular Biology: 25:12-18                              
   
Kurtzman, C.P. (2006). Yeast Systematio and Phytogeny. Implication of Molecular
                Identification Method in Ecology. 32:10-41

Nelson, M.D., John, Y.F. and Grustg, I.H. (2000). Microbiology of Yeast Diversity.           
           Cambridge: Harvard University Press.                       

Prentice, C.C. (1991). Concepts of Functional Biochemistry. Retrieved 6 July, 2010 from                      
        http://www.google.com


Rastall, G.B., Raphael, J.N. and Godwin, B.S. (1998). Microbial Innovation of Yeast  
              and fungal. Microbiology Science. Italy: Bioversity International-Headquater,
               P (274).

Reed, G.O.D., Pascal, B.M. and Nagodawithane A. (1991). Yeast Technology :( 2nd)
        New York: Van Nostrianl Rein hold.








APPENDIX A
Fiq1         RESULT FOR WEIGHT OF CARBON- DIOXIDE (CO2)
S/N
INITIAL(Y)
FINAL(X)
INITIAL – FINAL(Y-X)
WEIGHT OF CO (K)
1
641.4g
639.3g
641.4 - 639.3g
2.1g
2
654.1g
651.9g
654.1- 651.9g
2.2g
3
623.8g
621.4g
623.8- 621.4g
2.4g
4
629.0g
626.6g
629.0- 626.6g
2.4g
5
625.7g
623.3g
625.7 - 623.3g
2.2g
6
625.2g
622.7g
625.2 - 622.7g
2.8g
7
650.8g
648.4g
650.8 - 648.4g
2.4g
8
622.2g
620.0g
622.2 - 620.0g
2.2g
9
618.7g
616.6g
618.7 - 616.6g
2.1g
10
617.0g
614.8g
617.0 - 614.8g
2.2g
11
663.4g
661.7g
663.4 - 661.7g
1.7g
12
632.1g
629.7g
632.1- 629.7g
2.4g
13
636.6g
634.5g
636.6 - 634.5g
2.1g

% weight of carbon = initial weight – final weight.
The result of table 1 shows that the weight of the carbon dioxide present in each sample varies with respect to it level of fermentation.
Working expression
Initial weight (Y) – final weight (X) = weight of co (K)


APPENDIX B
MATHEMATICAL CALCULATION FOR VOLUME OF CARBON-DIOXIDE
SAMPLE A:
Weight = 2.1g
Density = 1.98g/ml
Volume = ?
Volume = 2.1g ÷ 1.98g/ml = 1.06ml
SAMPLE B:
Density = 1.98g/ml
Volume =?
Weight = 2.2g
Volume = 2.2g ÷ 1.98g/ml = 1.11ml
SAMPLE C
Density = 1.98g/ml
Volume =?
Weight = 2.4g
Volume = 2.4g ÷ 1.98g/ml = 1.21ml
SAMPLE D
Density = 1.98g/ml
Volume =?
Weight = 2.4g
Volume = 2.4g ÷ 1.98g/ml = 1.21ml
SAMPLE E
Density = 1.98g/ml
Volume = ?
Weight = 2.4g
Volume = 2.4g ÷ 1.98g/ml = 1.21ml
SAMPLE F
Density = 1.98g/ml
Volume = ?
Weight = 2.8g
Volume =2.8g ÷ 1.98g/ml = 1.41ml
SAMPLE G
Density = 1.98g/ml
Volume =?
Weight = 2.4g
Volume = 2.4g ÷ 1.98g/ml = 1.21ml
SAMPLE H
Density = 1.98g/ml
Volume =?
Weight = 2.2g
Volume = 2.2g ÷ 1.98g/ml = 1.11ml
SAMPLE I
Weight = 2.1g
Density = 1.98g/ml
Volume =?
Volume = 2.1g ÷ 1.98g/ml = 1.06ml
SAMPLE J
Density = 1.98g/ml
Volume =?
Weight = 2.2g
Volume = 2.2g ÷ 1.98g/ml = 1.11ml

SAMPLE K
Density = 1.98g/ml
Volume =?
Weight = 1.7g
Volume = 1.7g ÷ 1.98g/ml = 0.86ml
SAMPLE L
Density = 1.98g/ml
Volume =?
Weight = 2.4g
Volume = 2.4g ÷ 1.98g/ml = 1.21ml
SAMPLES M
Weight = 2.1g
Density = 1.98g/ml
Volume =?
Volume = 2.1g ÷ 1.98g/ml = 1.06ml

Fiq.2              RESULT FOR REVOLUME OF CARBON
S/N
SAMPLES
CONSTANT DENSITY (D)
WEIGHT (W)
VOLUME (W/D)
1
A
1.98g/ml
2.1g
1.06ml
2
B
1.98g/ml
2.2g
1.11ml
3
C
1.98g/ml
2.4g
1.21ml
4
D
1.98g/ml
2.4g
1.21ml
5
E
1.98g/ml
2.2g
1.21ml
6
F
1.98g/ml
2.8g
1.41ml
7
G
1.98g/ml
2.4g
1.21ml
8
H
1.98g/ml
2.2g
1.11ml
9
I
1.98g/ml
2.1g
1.06ml
10
J
1.98g/ml
2.2g
1.11ml
11
K
1.98g/ml
1.7g
0.86ml
12
L
1.98g/ml
2.4g
1.21ml
13
M
1.98g/ml
2.1g
1.06ml

The result in the Fiq 1 of the above table shows the volume of carbon-dioxide in each sample in relationship with the weight of the samples over the constant density of carbon dioxide.
Working expression
Volume (v) = weight (w) ÷ density (D).















APPENDIX C

Fiq 3.       RESULT FOR SUGAR LEVEL OF SAMPLES
S/N
SAMPLES
CONTROL
1ST DAY
2ND DAY
3RD DAY
4TH DAY
5TH DAY
6TH DAY
1
A
7
6
5
3
3
3
3
2
B
7
6
5
3
3
3.5
3.9
3
C
7
6
5
3
3
3
3.5
4
D
7
6
5
3
3
3
3
5
E
7
6
5
3
3
3
3
6
F
7
6
5
3.5
3
3
3
7
G
7
6
5
3
2.5
3
3.5
8
H
7
6
5
3
4
3
2.5
9
I
7
6
5
3.5
3
3
3
10
J
7
6
5
3.5
3.5
3.5
2.9
11
K
7
6
5
3
3.5
3
3
12
L
7
6
5
3
3
4
3.5
13
M
7
6
5
3.5
3.5
4
3.5

The result in fiq3 shows the decreasing level of the sugar present in each sample with respect to the days in which the sample undergoes fermentation, and the control (7) which was used as a constant for all the samples. This result was analyzed by the use of refractometer.