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.
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