Physicochemical, functional and microbial properties of crude and processed gum arabic (Acacia senegal) By Mahasin Elamin Mohammed Kheir B.Sc (Science) 1982 Faculty of Science, University of Khartoum A thesis submitted to the University of Khartoum in partial fulfillment for the requirements of the degree of Master of Science (Agriculture) Supervisor: Dr. Khogali Elnour Ahmed Department of Food Science and Technology Faculty of Agriculture University of Khartoum July 2005
Transcript
Physicochemical, functional and microbial properties of crude and processed gum
arabic (Acacia senegal)
By
Mahasin Elamin Mohammed Kheir B.Sc (Science) 1982
Faculty of Science, University of Khartoum
A thesis submitted to the University of Khartoum in partial fulfillment for the requirements of the degree
of Master of Science (Agriculture)
Supervisor:
Dr. Khogali Elnour Ahmed
Department of Food Science and Technology Faculty of Agriculture
University of Khartoum July 2005
DEDICATION
To my parents soles
HAJ : ELAMIN
HAJA : HAWAA
CONTENTS
Page ACKNOWLEDGEMENTS……………………………………........... i ABSTRACT…………………………………………………….......... ii ARABIC ABSTRACT……………………………………………….. iv CONTENTS…………………………………………………….......... vi LIST OF TABLES…………………………………………………………
4.2 Solubility and moisture content of gum arabic formulations........
33
4.3 Specific optical rotation, viscosity, acid insoluble ash and pH......
44
4.3.1 Specific optical rotation............................................................
44
4.3.2 Relative and intrinsic viscosity.................................................
44
4.3.3 Acid insoluble ash and pH.......................................................
44
4.4 Chemical constituents of the different gum arabic (Acacia senegal) formulations.......................................................................
ذي يدل على نقاء الصمغ اظهر بعض االختالفات بين الدوران الضوئي وال.بالرذاذ
درجة للمستحضر المجفف رذاذي 31.63- المستحضرات المختلفة والتي تتراوح مابين
.درجة لمستحضر القصة24.50 - درجة للنقاوة بينما وجد 31.28- و
فة، اللزوجة النسبية لم تظهر اختالفات معنوية بين المستحضرات الثمانية المختل
بينما اللزوجة الضمنية أظهرت اختالفات معنوية وكانت أعالها في مجروش حبيبات
أما بالنسبة . cm-3g-1)11.49 (119واقلها في مجروش حبيبات ) 18.85 (105
للمستحضر المجفف رذاذي % 0.09 الغير ذائب في الحمض تراوح مابين لمحتوى الرماد
.4022كان المتوسط لألس الهيدروجيني . للقُصة% 0.37و
% 3.05 و 121للمجروش حبيبات % 3.91تراوحت نسبة الرماد مابين
وة و للمستحضر النقا%0.42يتروجين فتراوحت نسبته بين أما الن. للمجفف بالرذاذ
بينما السكريات المختزلة، حامض اليرونيك والوزن المكافئ . للمجفف رذاذي0.37%
، %1.07وكانت قيمتها . تسجل اختالفات معنوية بين المستحضرات المختلفةوالجزيئي لم
. كمتوسط على التوالي1439.25و% 13.53
توجد اختالفات معنوية بين المستحضرات الثمانية المختلفة في المقدرة على حفظ
واألقل في %) 69.51( األعلى سجلت في المجفف رذاذي – كخاصية وظيفية–الماء
).%66.43 (105حضر مجروش حبيبات مست
. للقصة1.00 للمجفف رذاذي و 1.03بين الثبات االستحالبي تراوح
أظهرت الدراسة فروقات معنوية عالية بين المستحضرات الثمانية في العد
cfu/102×1.33 للمستحضر النقاوة و104cfu /gm×1.73البكتيري تراوحت مابين gm تم رصد .ضر المجفف رذاذي لم يسجل أي عد بكتيريالمستحبينما . 119للحبيبات
لمجفف الرذاذي، إضافة لخلو ا cfu/gm 10>الفطر في كل المستحضرات وقد كان
والسالمونيال في أي من المستحضرات اكل من الخميرة لم تظهر الدراسة وجود . منه
. الثمانيةلمختلفة من القيم المتحصل عليها للصفات والخصائص ا% 96كذلك وجد أن
.متناسقة مع المواصفات المطلوبة للصمغ العربي
CHAPTER ONE
INTRODUCTION
The oldest and best known of all natural gums is gum arabic obtained
from Acacia senegal, var. senegal. It is known as an important article
of commerce for about 4000 years ago. As time and technology
advanced, gum arabic is making a bigger and better place for itself
due to its relatively low prices and quality control assurance provided
by growing technology, so no artificial substituents match it for
quality or cost of production (Gabb, 1997).
The Sudan has been and is still the single largest producer of gum
arabic, the country supplies about 75% of the world needs of gum
arabic, (Gabb, 1997). Karamalla, et al, (1998).
Gum arabic is dried exudates obtained from the stems and branches of
Acacia senegal trees (Gabb, 1997). Chemically gum arabic was
defined as arabinoglactan-protein complex, proteoglycan acidic salt
(mainly Ca, K, Mg and Na) of high molecular weight (Gabb, 1997).
Polysaccharide mainly is composed of D-galactose, L-arabinose, L-
rhamnose, D-glucuronic acid and its 4-o methyl-ether together with a
proteinoceous component. The protein is an integral part of the
molecular structure of the gum arabic (Anderson et al., 1983), (Gabb,
1997).
Gum arabic is unique among the natural hydrocolloids because of its
extremely high solubility in water, forms viscous solutions up to 60%.
Quality means identity and purity of a certain product. Therefore, for
each product there are certain qualifying indices or parameters that
should be assured before the use or application.
The aim of this work could be summarized as follows:
Determination of the physicochemical and functional properties as
well as microbial load for raw and processed gum arabic produced in
the Sudan in an attempt to standardize specification and insure high
quality of the gum of Acacia senegal.
CHAPTER TWO
LITERATURE REVIEW
2.1 Distribution of Acacia senegal
About 500 species of Acacia are distributed over tropical and sub
tropical area of Africa, India, Australia and America but only a
comparatively few are commercially important. The important
producing areas are the Sudan, followed by Senegal, Mauritania, Mali
and Nigeria (Whistler and Bemiller, 1973).
The Sudanese gum belt extends from latitude 10° to 18°N where the
habitat Acacia senegal are sandy soils - arid zones from latitude 13°N
up words (Gum Arabic Company,G.A.C., 1993).
Gum arabic production in the Sudan is a traditional skill that has
evolved over many generations; always an important part of life in the
Sudanese gum belt, but to day has ever more economic and social
prominence (Gabb, 1997).
Gum production is evolving from tapping in the wild to a scientific
agro foresting operation providing higher and more reliable yields as
well as better quality of production (Gabb, 1997). Major markets for
gum arabic are the United States, United Kingdom, Italy, Germany,
Japan, France, Belgium and the Netherlands.
2.2 Gum exudation
There have been many theories concerning the phenomenon of
gummosis, but none of them is authenticated. (Whistler and Bemiller,
1973). Many authorities believe that the formation of gum exudates is
a pathological condition resulting from a microbial (fungal or
bacterial) infection of the injured tree. Edmonds (1965) and Anderson
and Herbich (1963), suggested that exudates come from natural
factors that tend to lessen the viability of the trees, such as poor soil,
hot weather and lack of moisture, which improve gum yields.
Anderson and Stoddart (1966) and Anderson and Dea (1971),
maintained the idea that the trees produce gum to seal the wounds and
prevent loss of water. They suggested that the gum produced by
Acacia is chemically and structurally related to the pheumococcus
polysaccharides, which encapsulate and protect the organism. Local
producers believe that a certain insect named locally Garraha is a
predisposing agent of gum production.
However, recent provisional data indicate that gum formation is not
related to this insect (Karamalla et al., 1998).
2.3 Acacia senegal gum
2.3.1 Description
Gum arabic is a pale white to orange–brown solid, which breaks with
a glassy fracture. The best grades are in the form of spheroid tears of
varying size with a matt surface texture, when ground, the pieces are
paler and have a glassy appearance. (JECFA, 1999).
Gum arabic is also available commercially in the form of white to
yellowish white flakes, granules powder; roller dried or spray dried
material. JECFA (1999).
2.3.2 Grades and processing:
The cleaned, hand picked selected grades (bigger tears, lighter color)
remain the qualities of choice for food, beverage and pharmaceutical
applicative (Joseleu and Ullmann, 1990), by kibbling, granulating,
powdering and spray drying.
2.3.3 Main uses:
The use of gum arabic to obtain superior quality in many products has
became so accepted in certain foods .It is an essential element in many
industries pharmaceutical, medicine, cosmetics, in local medicinal and
other industries.
2.3.3.1 Food industry
Gum arabic's emulsifying and film forming properties, low viscosity,
high solubility and stability in acidic media render it useful in many
applications within the food and flavor industry. (Glicksman, 1979-
Elizalde et al., 1988).
The important functional properties of gum arabic determine the range
of applications of such product into foods, is related to its interaction
with water, its ability to hydrate, swell and solublize, in addition
studies on water and oil absorption gum might explain its
effectiveness in emulsion stability.(Elizalde et al., 1988).
In addition, gum arabic is acceptable dietary intake (ADI) (JECFA
and EEC, 1999); non-toxic, odorless, colorless, tasteless, so it does not
affect the flavor, color or odor of the food to which it is added. In food
products gum arabic is used as a functional ingredient, which means
that the typical function of gum arabic, are emulsifier, flavoring
stabilizer and retards sugar crystallization.
The food applications of gum arabic have been developed from its
unequal combination of properties, emulsification, acid stability, low
viscosity at high concentration; adhesive and binding properties and
good mouth feel characteristics have been used in four main food area
worlds wide in descending order of importance. (Glicksmann, 1973).
2.3.3.1.1 Confectionary
Gum arabic is used to retard crystallization of sugar and to act as
emulsifier and as stabilizer in frozen dairy products, such as ice cream
and ices because of its water absorbing properties (Karamalla, 1999).
2.3.3.1.2 Beverages and emulsion
Gum arabic acts as oil and water emulsion stabilizer; it is a film
forming agent preventing coalescence of the oil globules.
2.3.3.1.3 Flavor encapsulation
Gum arabic is an ideal carrier in flavor encapsulation because of its
natural emulsifying and surface-active properties, good retention of
volatile flavor.
2.3.3.1.4 Bakery
Gum Arabic is used in bakery due to its viscosity, adhesive and
comparatively water absorption properties.
Gum arabic acts as foam stabilizer and colloidal agent in beer and
other beverages and as film forming on oily surfaces in chocolate and
snacks. Its low level of destabilization, high fiber content is useful in
diabetic and dietetic products (Whistler and Bemiller., 1973).
2.3.3.2 Pharmaceutical industry
Probably no more than 5% of the gum arabic is used for
pharmaceutical purposes. Its inherent emulsifying and stabilizing
properties, in addition to its demulcent and emollient characteristics
have led to a number of applications, ranging from the stabilizations
of emulsions to the preparation of tablets (Alain,.and McMullen,
1985). Its applications are further extended because it retains its
viscosity and stabilizing properties over a wide pH range.
Gum arabic is listed in British Pharmacopoeia (1993), as an effective
suspending acid and has been employed to suspend insoluble drugs
and to prevent the precipitation of heavy metals from solution through
the formation of colloidal suspensions (Whistler and Bemiller, 1973).
2.3.3.2.1 Medicine
Human dietary intake studies have indicated a reduction in blood
cholesterol levels when above average amounts of gum arabic
(25grams/day) are ingested in solution. The addition of a 7% gum
arabic solution reduces the dissipation rate of the sodium chloride
solution (Whistler and Bemiller, 1973).
2.3.3.2.2 Cosmetics
In lotions and protective creams, gum arabic stabilizes emulsions
increases the viscosity, adds smooth feel to the skin and forms a
protective coating used as an adhesive and constricting for facial
masks and face powder (Alain,.and McMullen, 1985).
2.3.4 Physicochemical properties of Acacia senegal gum:
2.3.4.1 Moisture
Loss on drying of good quality gum does not exceed 15% for granular
and 10% for spray-dried material (FAO, 1999), so moisture content
determines the hardness of the gum.
In a recent study reported by Karamalla et al., (1998) over 1500
authentic and commercial Acacia senegal var senegal, the results of
that study indicate the mean value of the moisture content as 10.75%.
Siddig (1996) reported that the range of moisture content for Acacia
senegal was 8.1-14.7%.
2.3.4.2 Ash
In a study of 800 authentic formulations of gum from Acacia senegal
var senegal collected from 32 different localities of the gum producing
belt of the Sudan, showed that the type of the soil had no significant
effect on the ash content of the gum (Karamalla et al., 1998).
Anderson et al., (1983) found that the value of ash content of
commercial gum arabic to be 4.4%.
Later, Anderson et al (1991) reported 3.6% Ash content for Sudanese
formulations. FAO (1999) reported that the ash content of gum arabic
did not exceed more than 4%.
2.3.4.3 Nitrogen and protein content
Gum arabic is a polymer with about 3% protein (Anderson et al.,
1991). The protein fraction is responsible for the emulsification
properties of the gum. The role of nitrogen and nitrogenous
component in the structure, physicochemical properties and
functionality of gum arabic was recently subjected to intensive
investigation. Structurally the "Wattle blossom" model (Fincher et al.,
1983) depends on the nitrogenous component (Fig.1).
An adsorbed layer of protein at oil /water interface provides the
primary stabilizing structure in many food colloids (Dickinson, 1994).
According to Dickinson et al., (1988), the variability in the
emulsifying properties of gums from different Acacia species is
dependent not only on the total protein (on polypeptide content) but
also on the distribution of the protein-peptide between the low and
high molecular weight fractions and on the molecular accessibility of
the protein peptide for absorption according to Randall et al., (1989).
The United States pharmacopoeia and European Union specification
defined the minimum standards of the protein content for good quality
gum arabic as 3%.
Anderson (1986) found that the average nitrogen content for
commercial Acacia senegal gum formulations to be 0.37%.
Arabinogalactan substituent
Polypeptide backbone
Protein – polysaccharide Linkage region
Figure (1) The Wattle – Blossom model for Acacia senegal gum as proposed by Fincher et al.,(1983 ) .
Investigations of protein in Acacia senegal gum have been carried out
by (Akiyama et al., 1984), they reported that gum arabic contained
2.0% protein and they established that amino acid of gum arabic is
rich in hydroxyproline and serine while alanine content is low.
Anderson et al., (1985) described gum arabic as a proteinaceous
polysaccharide with a protein content ranging from 1.5 to 3.0%. And
concluded that the variation was mainly due to different localities.
They reported the value of 0.23-0.58% nitrogen for commercial
formulations. Osman (1998) reported 0.33- 0.36% nitrogen (2.14-
2.16% protein for Acacia senegal gum) and Jurasak et al., (1993) in a
chemo metric study for different Acacia species reported 0.27-038%
nitrogen for commercial samples of Gum arabic from Sudan. Awad
Elkarium (1994) reported that the average nitrogen contents of
different commercial grades are around 0.28%. Karamalla et al.,
(1998) reported that the average nitrogen content of different
commercial grades is around 0.33%.
2.3.4.4 Acidity and pH
Comparative studies Show that gum from Acacia senegal has higher
content of rhamnose (12-14%) and lower arabinose content (24-29%)
compared to rhamnose and arabinose of other Acacias (Karamalla et
al., 1998).
The main content of gum arabic is arabian (acid substance), and when
it was decomposed it gave arabinose (Mantell, 1965). Thus gum
arabic is called arabic acid therefore, the gum solution is slightly
acidic with pH 4.66, as reported by Karamalla et al., 1998.
2.3.4.5 Specific optical rotation
Acacia senegal gum is the highest quality compared to other gums
species. The problem is that differentiating between the species, once
the gum is processed is difficult. The specific optical rotation is
considered as the most important criterion of purity and identity of
gum arabic, so it is used to differentiate between Acacia senegal gum
and other botanically related Acacia gums.
FAO (1990) considered that the specific optical rotation of Acacia
senegal gum to be ranging between -26° to -34°. Recently Karamalla
et al., (1998) showed that the mean value of specific rotation of the
commercial Acacia senegal gum was -31°.
2.3.4.5 Solubility
Gum arabic is unique among the natural hydrocolloids because of its
extremely high solubility in water and can yield solutions of up to
60% concentration and it is truly soluble in cold water, other gums
are either insoluble in cold water or form colloidal suspensions “not
true solutions” (G.A.C, 1993).
2.3.4.6 Equivalent weight and Uronic Acid
Titrable acidity represents the acid equivalent weight of the gum, from
which the uronic acid content could be determined (Karamalla, 1965,
Anderson et al., 1983).
Karamalla et al., (1998), assessed the potentials of new parameters
such as equivalent weight and total uronic acid content as additional
qualifying indices. They found that the mean values for gum of Acacia
senegal for the equivalent weight was 1436 and for uronic acid was
13.71%.
2.3.4.7 Viscosity
(Most gum other than gum arabic form highly viscous solutions at low
concentrations of 1.5%). Gum arabic unique in that it is extremely
soluble in water and is not very viscous at high concentrations. High
viscosities are not obtained with gum arabic until concentrations of
about 40-50% are obtained, (Sharma, 1979b). This ability to form
highly concentrated solutions is responsible for the excellent
stabilizing and emulsifying properties of gum arabic when
incorporated with large amounts of insoluble maters. (The viscosity of
gum solutions will depend upon the type and variety used). At
concentrations up to 40% gum arabic exhibit typical Newtonian
behavior. At about 40%, solutions took up pseudo plastic
characteristics as denoted by decrease in viscosity with increasing
shearing stress (Sharma, 1979a).
Studies of flow of gum solution play an important role in
identification and characterization of their molecular structure.
Kaufman and Falcetta (1977) showed that viscosity could be
presented in many terms such as relative viscosity, specific viscosity,
reduced viscosity and intrinsic viscosity.
2.3.4.8 Intrinsic viscosity
It can be used to determine the molecular weight of Acacia senegal
gum (Anderson and Dea, 1971).
Karamalla, (1999) showed that wide variations in values for intrinsic
viscosity and viscosity average were obtained indicating that such
parameters cannot be used as qualifying Indices.
2.3.4.9 Molecular weight
Gum from Acacia senegal was reported to have high molecular weight
average 4×105 Mv. (Anderson et al., 1983). The most common
method used to determine the molecular weight was the intrinsic
viscosity measurements (Anderson et al., 1983 and Phillips and
Williams., 1988).
Application of Mark-Houwink equation indicated that the average
molecular weight of Acacia senegal gum was 4.6×105 (Alamin and
Norbest., 1985). However, Eric et al., (1991) reported an average of
2.2×105 to 3.1×105 for molecular weight of degraded products of gum
arabic. Moreover, Karamalla et al., (1998).Found the molecular
weight of Acacia senegal to be 4×105 Mv.
2.3.4.10 Reducing sugars
The presence of reducing sugars would give evidence to the reducing
power (free reducing groups) of the gum (Somogi, 1945). It is usually
calculated as arabinose.
Anderson and Karamalla. (1966) reported 0.16-0.44% as the range
value of reducing sugar for Acacia senegal gum.
2.3.5 Functional properties:
2.3.5.1 Emulsifying stability
Gum arabic is a very effective emulsifying agent because of its
protective colloid functionality and has found wide use in the
preparation of varied oil-in water food emulsions with most oils over a
wide PH range and in the presence of electrolytes without the need for
a secondary stabilizing agent .( Phillips and Williams., 1988).
Emulsifiers are classified as a group of surface-active agents that can
stabilize a dispersion of two liquids such as water and oil, which are
essential for emulsion formation and stabilization to occur (Kinsella,
1979).
2.3.5.2 Water holding capacity (W.H.C)
Is the ability of the material to hold water against gravity (Hansen,
1978).
The range value of water holding capacity reported by Omer (2001)
for Acacia senegal gum was 65.40-65.80.
2.3.5.3 Encapsulating agent
Microencapsulating is a process where droplets of liquids, solids, or
gases (core) are coated by thin film (coatings) e.g. gum arabic, which
protects the core until it is needed (Joseleau and Ullmann1990). The
coating on a core is semi-permeable and protects the core from severe
conditions and controls substances flavoring into the core.
The major use for encapsulation in food industry is for liquid flavors.
Encapsulation has been able to mask bitter tastes of compounds,
reduce volatility and flammability of liquids, control release of
materials, provide protection to compounds, reduce toxicity, separate
reactive materials and to make liquids behave like solid.
Micro encapsulation by spray drying is the most economical and
flexible way for the food ingredient to retain the needed properties in
the final food products.
This technique also has been used in pharmaceutical industries e.g.,
vitamins and minerals (Joseleau and Ullmann1990). Yet is not widely
used in the food industry.
2.3.6 Gum arabic microbiology
Gum arabic has been subjected to extensive research on its chemical,
physical, and functional properties aiming at setting definite criteria
for quality control. However, little attention has been given to the
microbiological aspects of this very important commodity.
Studies of about fifty-two formulations of authenticated and
commercial Acacia senegal gum (raw and processing gum arabic)
were tested for the count of total bacteria, mould, yeasts and coli
forms. The maximum microbial loads of these formulations were
about 3×105cfu/gm bacteria, and 1.6×104cfu/gm of moulds. The
microbial load of processed gum formulations were much lower than
the loads of raw gum one, and ranged between Nil to a maximum of
<10 cfu/gm. Salmonella and yeasts were not detected in any of the
formulations tested (Karamalla., 1999).
A comparative microbiological study on authenticated and
commercial Acacia senegal gum formulations obtained from various
locations in the Sudan showed that gum arabic harbored considerable
numbers of bacteria and fungi (Osman, 1998). However, no
microbiological examination of gum arabic has hitherto showed the
presence of pathogenic microorganisms. Analytical data provided by
Vollard (1972) on different formulations of gum arabic (both nodules
and in powdered form) showed that gum arabic was in general, of
acceptable microbiological examination of industrially processed.
Sudanese Acacia senegal gum confirmed the freeness of raw or
processed gum arabic from pathogenic bacteria (Khalid et al., 1988).
Idris (1986) and Osman (1998) obtained similar conclusions.
Accordingly, gum arabic is officially classified by the joint
FAO/WHO expert committee on food additives (JECFA, 1999) as a
“safe and acceptable dietary intake” (ADI).
The dominant bacteria contaminating gum arabic formulations were
gram (+ve) rod shaped, spore-forming ones. Gram (+ve) non-spore
forming rods and gram (+ve) cocci types of bacteria were also
detected, but insignificant amounts. (Karamalla et al., 1998).
An experiment carried out to simulate the effect of sunlight and UV
light on microbial load of formulations resulted in a remarkable
decrease in the bacteria and mould counts.
Although the microbial counts were well within the acceptable limits,
efforts have been made to develop a simple technique to reduce such
counts (Karamalla et al., 1998). The proposed application of
UV/radiation may lead to the possibility of alterations in the
carbohydrate composition of the gum. The possibility of changes in
some of the physical characteristics (gummosis) due to elimination of
some of the natural micro flora cannot be ignored (Karamalla et al.,
1998). Studies clearly indicated that processing reduces microbial
counts of the gum arabic; therefore reducing the moisture content of
the natural gum can be readily used as a tenable method of reducing
the microbial count (Karamalla et al., 1998).
CHAPTER THREE
MATERIALS AND METHODS
3.1 Materials:
3.1.1 Gum arabic formulations:
Eight formulations of crude and processed gum arabic were collected
for analysis, they were:
A1, A2, A3, A4, A5, A6, A7 and A8.
They can be classified as follows:
3.1.1.1 Raw gum arabic, which included:
A1: Hand picked selected.
A2: Cleaned.
A3: Sifting.
These were supplied by gum arabic company (GAC).
3.1.1.2 Kibbled gum arabic, which included:
A4: kibbled 107 of mesh <6mm.
A5: kibbled 121 of mesh<210micro.
A6: kibbled 119 of mesh<1mm.
A7: kibbled dust
These were obtained from gum arabic processing company (GAPC)
3.1.1.3 Spray-dried gum:
It is sample A8. Obtained from Gaby Haddad Company.
3.1.2 Growth media
1. Malt extract agar was used for the isolation, counting and
cultivation of moulds and yeasts.
2. Rappaport-Vassiliadis medium was used as enrichment for
salmonella.
3. Xylose, lactose, deoxycholate agar (XLD). Solid selective agar
for salmonella.
4. Buffered peptone water was used as resuscitation for salmonella
and moulds and yeasts.
5. Tetracyanate media.
6. Plate count agar for total bacterial count.
7. Bismuth sulphite. Solid selective agar for salmonella.
3.1.3 Preparation of formulations
The gum arabic formulations used in this study were supplied by
exporting companies from the products prepared for exportation.
Then the formulations of hand picked selected (HPS), kibbled,
cleaned and sifting were ground using mortar and pestle 1100 until the
standard finest powder of mesh size No. 10 was obtained.
3.2 Analytical methods
Some physicochemical, functional and microbial loads for
formulations were determined to evaluate existing quality parameters.
The following analytical methods were adopted for the study.
3.2.1 Physicochemical analysis
3.2.1.1 Solubility
Solubility was obtained by dissolving 1g of formulation (W) in 100ml
of each of the following solvents, distilled water, ethanol, chloroform
and acetone in 250ml conical flask, then stirred for 30 minutes by
magnetic stirrer and the solution filtered through filter paper No. 41,
which was weighed before filtration (W1). The filter paper and
contents were then dried at 105°C for 30 minutes, cooled and weighed
(W2).
The solubility was calculated as percent according to the following
equation:
S% = W – (W2 – W1) W Where:
S = Solubility
W = Weight of formulation
W1 =Weight of empty filter paper
W2 = Weight of filter paper + insoluble formulation
3.2.1.2 Moisture content
According to FAO (1990), the determination was conducted on
accurately weighed, 2g of previously well-mixed gum powder.
An empty crucible was dried in Herateus oven at 105°C for 30
minutes, cooled in a desicator and weighed (W1), about two grams of
the formulation were placed in the crucible and weighed accurately
(W2) and heated for 5 hours at 105°C, cooled in a desicator and
weighed again (W3). The loss on drying was calculated as follows:
(W2-W3) × 100
(W2-W1)
Where:
W1 = Weight of the empty crucible
W2 = Weight of the crucible + formulation
W3 = Weight of the crucible + formulation after drying.
3.2.1.3 Nitrogen and protein contents
Nitrogen and crude protein were determined using a semi-micro
Kjeldal digestion and distillation method according to AOAC (1984).
1g of the formulation was weighed into 100ml Kejeldal flask, 2 tablets
of catalyst mixture (potassium sulfate + copper sulfate) and 12.5ml
concentrated sulfuric acid were added. The flask containing all these
mixtures was heated on an electric heater for digestion for two hours,
then was cooled and placed in the distillation unit. The ammonia
evolved was received in 25ml of boric acid solution containing 3
drops of mixed indicator (bromocresol, green and methyl red).
The trapped ammonia was titrated against 0.1N HCL, hence the
protein percentage was determined according to Anderson (1986), by
multiplying nitrogen percentage N% by the factor 6.6.
N% = V × N × 14 × 100 S Where:
V = Volume of HCL
14 = Atomic mass of nitrogen
N = Normality of HCL (mol / dm3)
S = Weight of sample.
Protein % = N% × 6.6
Where:
6.6 = the nitrogen factor for gum arabic as proposed by Anderson
(1986).
3.2.1.4 Total ash content
The ash percentage was determined according to FAO (1990), a
crucible was heated at 105°c for 30 minutes, cooled in a desicator and
weighed (W1), about 2g of formulation were accurately weighed in a
crucible (W2), and ignited in Heraeus electronic muffle furnace at
550°C until free from carbon, cooled in a desicator and weighed (W3),
then the total ash percentage was calculated as follows.
W2 – W3 ×100
W2 – W1
Where:
W1 = Weight of the empty crucible
W2 = Weight of the crucible + the formulation
W3 = Weight of the crucible + ash
3.2.1.5 Specific optical rotation
The specific optical rotation was determined for 1.0 % aqueous
solution on dry weight basis using an optical activity Bellingham and
Stanley ltd.AD 220 polar meter fitted with sodium lamp and with a
cell path length of 20 decimeter at room temperature (25°c) after
filtration of the gum solution through filter paper No. 42.
Readings were taken three times and averaged. The specific optical
rotation was calculated according to (Moflit and Young, 1956) using
the following equation:
[α]td = Z × 100
C × L
Where:
α = Specific optical rotation
Z = observed optical rotation
C = concentration of solution
L = Length of polar meter cell in decimeter
D = Sodium lamp λ = 589 nano meter (nm.)
3.2.1.6 pH value
pH was determined in 10% aqueous solution using Ion Meter 3205
JENWAY.
Two standard buffer solutions of pH 4.00 and 7.00 were used for the
calibration of the pH meter. The temperature was kept at 25°C and the
pH was let to stabilize for one minute and then the pH of gum
formulations were read directly. The readings were repeated three
times for each formulation.
3.2.1.7 Molecular weight
The molecular weight was calculated using Mark-Houwink equation
(Mark, 1938., Houwink, 1940).
(η) = K × Mwa
Where:
Mw = Molecular weight
(η) = Intrinsic viscosity
K and a = Mark -Houwink constant
Based on (Anderson and Rahman, 1967), the values of K and a, were
determined for Acacia senegal gum as follows:
K = 1.3 × 10-2
a = 0.54
3.2.1.8 Tannin content
0.1 ml Ferric chloride was added to 10 ml 1% aqueous gum solution.
Presence of blackish coloration or precipitate indicates the presence of
tannin (FAO, 1999).
3.2.1.9 Acid insoluble ash
The ash was boiled with 25ml dilute hydrochloric acid TS for 5
minutes. The insoluble matter was collected on a suitable ash less
filter paper, then washed with hot water and ignited at 800°C ± 25°,
cooled and weighed, the percentage of acid insoluble ash was then
calculated from the weight of the formulation taken.
3.2.1.10 Viscosity measurement
Viscosity was measured using U-tube viscometer (type BS/ IP/ U,
Serial No. 2948) with the flow time for 1% aqueous solution of
formulation at room temperature (25°C). The relative viscosity (ηr)
was then calculated using the following equation:
ηr = T - To To Where:
T = flow time of formulation solution expressed in seconds.
To = flow time of solvent distilled water (DW) expressed in
seconds.
The reduced viscosity (ηrd) was determined for different
concentrations of gum solution 5, 10, 15 and 20% and was then
calculated from the following equation:
ηrd = ηr / C
Where:
ηrd = reduced viscosity
ηr = relative viscosity
C = concentration of formulation solution
3.2.1.11 Intrinsic viscosity (η)
The intrinsic viscosity was obtained by extrapolation of reduced
viscosity against concentrations back to zero concentration. The
interception on Y – axis gives (η).
2.2.1.12 Uronic Acid
Uronic acid percentage was determined according to Anderson et al
(1983) by multiplying the molecular weight of the uronic 194 by 100
and dividing by the apparent equivalent weight of the formulations as
follows:
Uronic acid % = 194 × 100 Equivalent weight
3.2.1.13 Reducing Sugars
Reducing sugars were determined according to Robyt and White
(1987), using alkaline Ferricyanide method. The procedure uses a
single reagent composed of 0.34g of potassium ferricyanide 5g of
potassium cyanide and 20g of sodium carbonate dissolved in 1 liter of
distilled water. 1.0ml of gum solution was added to 4.0ml of reagent,
then heated in a boiling water bath for 10 minutes and cooled. The
absorbance was measured at 420nm.
Standard curve was prepared from different arabinose concentrations
plotted against absorbance in order to calculate the reducing sugars
concentrations as arabinose (Fig.2).
3.2.1.14 Apparent Equivalent Weight
According to the method reported in the Encyclopedia of Chemical
Technology (1966) with some modifications. The aqueous gum
solution 3% was treated with Amberlite Resin (120) (H+) (2 grams per
10mls solution) then shaken for an hour and titrated against 0.02N
sodium hydroxide solution using phenolphthalein as indicator. The
equivalent weight was calculated as follows:
Eq. Wt = 50.000 ×0.3
M
Where:
Eq. Wt = Equivalent weight.
M = No. of mls of 0.02N sodium hydroxide neutralizing 10ml
of 3% formulation solution.
0.3 = No. of grams of gum per 10 ml of the 3%
solution
Standard Curve for Reducing Sugar Concentration (as arabinose % at 240 nm)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 0.25 0.5 0.75 1 1.25 1.5 1.75
Concentration %
Abs
orpt
ion
at 4
20 n
m
Figure (2) Standard Curve for Redusing Sugar Concentrations (as arabinose% at 420nm)
3.2.2 Functional Properties Analysis
Functionality analysis of Acacia senegal gum of different forms
(Hand picked selected, kibbled and spray dried) were carried out as
follows:
3.2.2.1 Water Holding Capacity (WHC)
It was determined according to Hansen, (1978). 1g of gum was
accurately weighed in a Petri-dish, then it was placed in desiccator
half-filled with distilled water and incubated for certain lengths of
time: 24, 48, 96, 120, and 144 hours. The Petri-dish with formulation
was then weighed (g/g) and finally expressed as percentage as
follows:
WHC% = Weight of water × 100
Weight of formulation
Where:
WHC = Water holding capacity.
3.2.2.2 Emulsifying Stability
It was determined according to Kinsella, (1979). Gum arabic solution
(20% concentration) was mixed with oil (sunflower oil) at a ratio of
80: 20 W/W, respectively. They were mixed using a blender for one
minute. The mixture was then diluted to a ratio of 1: 1000 and it was
read at λ max. 520nm.
The second reading was taken after one hour. The readings
represented emulsifying index.
Emulsifying stability (E.S.) was calculated as follows:
(E.S.) = First reading
Reading after 1 hour
3.2.3 Microbial Load
3.2.3.1 Determination of total bacterial count( pour plate count).
Figure (3) shows the steps followed for the determination of bacterial
count in gum arabic formulations according to testing methods in food
microbiology and manual of food quality control microbiological
analysis (Andrews, 1992).
10 g of gum arabic were suspended in 90ml sterile distilled water and
were shaken till completely dissolved to release any microorganisms
trapped inside and the suspension was left to equilibrate. From this
suspension 1/10, 1/100, and 1/1000 strength dilutions were prepared.
1.0ml of each of dilutions which prepared from the stock suspension
was pipetted into sterilized labeled Petri dish (the label should indicate
the formulation and degree of dilution), beginning with the highest
dilution.
About 15ml of melted medium (plate count agar) were poured into
each Petri-dish. The raised lid of the Petri dish is lowered again and
the suspension is cautiously mixed with the medium by a combination
of to and fro and circular movements over the horizontal surface of
bench. The plates were incubated at 37 ºC for 48 hours.
The count of bacteria was expressed as colony-forming units (cfu)
using a Scientific Colony Counter (Scientific and Cook Electronic
Ltd., England). Then multiplying the number of colonies by the
reciprocal of the dilution, expresses aerobic plate count as number of
organisms per gram of food.
Figure (3) Scheme for the steps of determination of bacterial plate
count in gum arabic formulations
450 ml of pepton water + 50 gm of formulation
10-2 10-3 10-4 10-5 10-6
3. Pipette 1ml volume into sterile plates
10-1
4. Pour 20 ml of plate count Agar.
5. Incubate at 37ºC for 48hours.
6. Calculation of the colonies.
1. Blend Preparation of homogenous formulation of the ratio 1: 9
2. Dilution
Preparation of different dilutions
3.2.3.2 Determination of moulds and yeasts
Figure (4) shows the steps followed for the determination of moulds
and yeasts in gum arabic formulations according to Harrigan, (1998).
25g of gum arabic were suspended in 225ml peptone water and were
shaken until completely dissolved to release any microorganisms
trapped inside, and the suspension was left to equilibrate. That
suspension was used to prepare the following dilutions:
1/10, 1/100 and 1/1000 strength. Then 0.1ml of each dilution was
surface-plated on a malt extract agar Petri dishes, which contained
chloramphenicol as an antibiotic. The Petri-dishes were then
incubated at 25ºC ±1º for five days. The last step was calculating the
number of colonies by multiplying the number of colonies by the
reciprocal of the dilution.
3.2.3.3 Determination of Salmonella
Figure (5) shows the steps followed for the determination of
Salmonella in gum arabic formulations according to Harrigan, (1998).
25g of gum arabic were suspended into 225ml of buffered pepton
water at PH 7.2 ± 0.2 and incubated at 37°Cfor 24 hours. 10 ml of the
suspension were mixed with 90ml of Rappaport broth and was
incubated at 37°C for 24 hours. 10ml of that mixture were mixed with
90ml of tetracyanate and were then incubated at 37°C for 24hours.
The culture was done by using a loop, from this mixture in the two
solid selective agar XLD and Bismuth sulphite separately, then
incubated at37°C for 24 hours.
Figure (4) Scheme for the steps of determination of moulds and
yeasts in gum arabic formulations
10-2 10-3 10-4 10-5 10-6
2. Preparation of Different dilutions in the ratio of 1:10
3. Pippete 0.1ml of the formulation into sterile dish
4. Pour 15ml of mould extract Agar with antibiotic
5. Incubation at 25ºC ± 1 for 30 hours
6. Calculation of the colonies.
225 ml pepton water +25 gm formulation
10-1
3. Incubation at 37°C for 24 hours
Figure (5) Scheme for the steps of determination of Salmonella in
gum arabic formulations
225 ml of pepton water 25 gm of the formulation
1. Preparation of homogenous formulation Incubation at 37°C for 24 hours
2. Incubation at 37°C for 24 hours
90 ml of rappaport broth 10 ml of formulation
90 ml of tetrathionate broth + 10 ml of formulation
Bismuth sulphite XLD
4. Incubation at 37°C for 24 hours
5. Calculation of the colonies
3.3 Statistical analysis The data on various parameters in the different gum arabic
formulations (GAF) were subjected to statistical analysis using SPSS
(Sincich, 1982) program Analysis of variance (ANOVA) were carried
out for that data based on the completely randomized design (CRD).
The confidence interval (at 95%) technique was adopted for
comparing the standards and specifications with the values of the
parameters under study.
To simplify the comparative studies the ranges of the specifications
and standards were averaged. Scoring system (Laoata, personal
communication) was advocated to come out with a conclusion
regarding the proportion of values of the parameters studied in GAF
that match with the standards.
CHAPTER FOUR
RESULTS
4.1 Physical properties shape, color and mesh of gum arabic
(Acacia senegal) formulations:
Table (1) illustrates the evaluation of the shape, color and mesh of the
eight gum arabic formulations. The formulations can be categorized
according to the shape into: spheroidal tears and/or angular fragments;
and powder. However these forms were different in size of the
particles or mesh, (Table 1). The hand picked selected and cleaned
gum formulations were in the natural form, color and size of the
particles. On the other hand, the kibbled and spray dried formulations
were passed through some processing steps; hence, their shape, color
and mesh were changed.
4.2 Solubility and moisture content of gum arabic formulations:
The solubility of the gum arabic formulations in water and some other
chemical solvents are shown in table 2 and figs. 6-12.
Highly significant differences were existed between the Gum Arabic
Formulations (GAF) in water solubility. The highest solubility was for
the spray dried (98.80±1.71%); and the lowest for the sifting
formulation (97.40±1.71%).
As for the other chemical solvents, the findings concerning GAF were
given in table 2. Highly significant differences between GAF in the
level of solubility in ethanol and chloroform were found. The
differences between the ethanol solubility of the formulation and in
acetone were also significant (at 5%). Very low solubility in ethanol,
acetone and chloroform could be inferred from table 2.
The moisture contents were significantly different between the eight
GAF and ranged between 9.90% in the hand picked selected and
8.32% in spray dried (Table 2).
Table (1) Evaluation of the shape, color and mesh of the eight gum
arabic (Acacia senegal) formulations.
Gum Arabic
Formulation Shape Color Mesh Hand picked Spheroidal tears Pale white to varying mesh selected orange –brown Cleaned Spheroidal tears White to varying mesh Yellowish-white Flakes Sifting Small particles orange-brown small particles with different sizes
Kibbled 107 Spheroidal tears. White to <6 mm or in angular Yellowish-white
Fragments Flakes Kibbled 121 Spheroidal tears. White to <210 micron or in angular Yellowish-white Fragments
Kibbled 119 Spheroidal tears. White to <1 mm or in angular Yellowish-white Fragments Kibbled 105 Spheroidal tears White to <8 mm or in angular Yellowish-white Fragments Spray-dried Powder White to <0.3mm Cream powder
Table (2) Solubility of the different Gum arabic (Acacia senegal) formulations in different solvents
Solubility in different solvents (%) Gum arabic
formulation Water Ethanol Acetone Chloroform
Moisture
content (%)
Hand picked
selected 97.59 5.00 9.00 13.00 9.90
Cleaned 97.54 3.50 7.20 10.40 9.44
Sifting 97.40 9.50 6.20 13.60 9.14
Kibbled 105 98.73 10.50 11.30 15.40 8.38
Kibbled 107 97.50 2.50 3.60 5.30 8.86
Kibbled 119 98.72 2.90 5.30 4.90 8.53
Kibbled 121 98.72 3.60 4.10 5.20 8.37
Spray- dried 98.80 1.50 1.20 1.40 8.32
Mean 98.01 5.00 5.98 8.65 8.87
SE (±) 1.71** 0.08** 2.23* 0.77** 1.06**
C.V. (%) 3.02 1.17 64.27 8.84 20.72
* = Significant differences at 5% level
** = Significant differences at 1% level
Figure (6) The solubility of the hand picked selected formulation in different solvents.
0
10
20
30
40
50
60
70
80
90
100
Water Ethanol Acetone Chloroform
Figure (7) The solubility of the cleaned formulation in different solvents.
0
10
20
30
40
50
60
70
80
90
100
Water Ethanol Acetone Chloroform
Figure (8) The solubility of the sifting formulation in different solvents.
0
10
20
30
40
50
60
70
80
90
100
Water Ethanol Acetone Chloroform
Figure (9) The solubility of the kibbled formulation in different solvents.
0
10
20
30
40
50
60
70
80
90
100
Water Ethanol Acetone Chloroform
Figure (10) The solubility of the kibbled 107 formulation in different solvents.
0
10
20
30
40
50
60
70
80
90
100
Water Ethanol Acetone Chloroform
Figure (11) The solubility of the kibbled 119 formulation in different solvents.
0
10
20
30
40
50
60
70
80
90
100
Water Ethanol Acetone Chloroform
Figure (12) The solubility of the spray dried formulation in different solvents.
0
10
20
30
40
50
60
70
80
90
100
Water Ethanol Acetone Chloroform
4.3 Specific optical rotation, viscosity, acid insoluble ash and pH
4.3.1 Specific optical rotation
Table 3 shows the specific optical rotations for the gum arabic
formulations. The highest value was recorded in the spray dried
formulation (-31.63±0.31 degrees), whereas the lowest in sifting
formulation (24.50±0.31 degrees).
4.3.2 Relative and intrinsic viscosity:
The relative and intrinsic viscosities of the eight GAF were shown in
table 3 and fig.13. Highly significant differences were noticed
between the formulations for the intrinsic viscosity; however, no
significant differences were noticed between GAF in the relative
viscosity.
4.3.3 Acid insoluble ash and pH:
Highly significant differences were found in the value of acid
insoluble ash between the different GAF (Table 3).
The lowest level of acid insoluble ash was in the spray dried whereas
the highest in sifting formulation (0.09 and 0.38; respectively).
The formulations showed significant variation in pH with an overall
average of 4.22 (acidic) table 3.
4.4 Chemical constituents of the different gum arabic (Acacia
senegal) formulations:
4.4.1 Ash (%)
Highly significant differences in the level of ash were detected
between the different formulations (Table 4) the overall mean of the
level of ash was 3.64.
The highest level of ash was in kibbled 121 (3.91 % ± 1.56) whereas
