ADDIS ABABA SCIENCE AND TECHNOLOGY UNIVERSITY

DEVELOPMENT AND EVALUATION OF PREBIOTIC

FOOD INGREDIENT USING GREEN BANANA PEEL,

CARROT BAGASSE AND ORANGE PEEL COMPOSITE

POWDER.

By

GEBEYEHU AYELE HIDO

A Thesis Submitted as a Partial Fulfillment to the Requirements for the Award of the Degree of

Master of Science in chemical engineering (Food Process Engineering)

to

DEPARTMENT OF CHEMICAL ENGINEERING

COLLEGE OF BIOLOGICAL AND CHEMICAL ENGINEERING

JANUARY, 2022
 Approval Page
This is to certify that the thesis prepared by Mr. Gebeyehu Ayele Hido entitled “Development
and Evaluation of Prebiotic Food Ingredient Using Green Banana Peel, Carrot Bagasse and
Orange Peel Composite Powder” and submitted as a partial fulfillment for the award of the
Degree of Master of Science in Chemical Engineering (Food Process Engineering) complies with
the regulations of the university and meets the accepted standards with respect to originality,
content and quality.

Signed by Examining Board:

Advisor: Signature, Date:

External Examiner: Signature, Date:

Internal Examiner: Signature, Date:

Chairperson: Signature, Date:

DGC Chairperson: Signature, Date:

College Dean/Associate Dean for GP: Signature, Date:

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 Declaration
I hereby declare that this thesis entitled “Development and Evaluation of Prebiotic Food
Ingredient Using Green Banana Peel, Carrot Bagasse and Orange Peel Composite Powder”
was prepared by me, with the guidance of my advisor. The work contained herein is my own except
where explicitly stated otherwise in the text, and that this work has not been submitted, in whole or
in part, for any other degree or professional qualification.

Author: Signature, Date:

Witnessed by:

Name of student advisor: Signature, Date:

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 ABSTRACT

The aim of this work was to develop and evaluate prebiotic food ingredient by using green banana
peel, carrot bagasse and orange peel composite powder and formulate the soup. Inadequate prebiotic
in the diet can lead to different health problems like less resistance of body to invading pathogens,
inadequate absorption of minerals, the body is easily attacked by different diseases such as cancer,
diabetes, respiratory diseases, increased blood level and etc. The individual powder was produced
from each raw material to formulate composite powder based on Box-Benken Experimental
Design. Green banana peel powder (20, 25, & 30 gram), carrot bagasse powder (30, 35 & 40 gram),
and orange peel powder (15, 20, & 25 gram) with three levels to formulate 18 different combinations
of composite powder, were studied to determine proximate composition, functional properties,
investigate the prebiotic properties, probiotic growth stimulation and oxidant capacity. The moisture
content, ash, crude fat, protein, and crude fiber of composite powder was in the range 3.1- 14.5%,
6.0- 18.2%, 2.0- 10.0%, 0.5- 7.0% and 84.2- 69.6% respectively. The functional properties of
composite ranged from 1.52- 4.35g/g, 7.02- 11.04g/g, 0.77-0.95g/g, and 18.20-23.42g/g for oil
holding capacity, water holding capacity, bulk density and swelling capacity respectively. According
to the research, composite powder was less vulnerable to acidic digestion regardless of incubation
period, the findings showed that it can be considered a viable prebiotic because it possesses prebiotic
features such as non-digestibility. The maximum resistivity of hydrolysis was 31% from the study.
Inulin extraction from composite powder with ethanol extraction was 2.34g from the sample 10. The
composite powder showed satisfactory growth stimulation in Lactobacillus acidophilus NCFM
which ranged from 6.98- 8.12 log10 CFU/Ml.

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 Acknowledgements
I would like to thank the almighty God for his help for everything and my families for their
genuine support till this time .I would like to build my deepest appreciation and gratitude thanks to

my advisor Dr. Venkatesa Prabhu.S (Assistant Prof.) for his valuable guidance, constructive

criticism and encouragement during every stage of this research paper. I would like to acknowledge
Addis Ababa Science and Technology University Department of Chemical Engineering for

providing the grant funding to this research. I am gratifying to thank Dr. Ali S. former Head of

Chemical Engineering Department for providing me the necessary opportunities for the completion
of my research paper. I would like to thank Dr. Girma Gonfa for his help in providing statistical
analysis tool for analyzing data. Also I would like thank Ethiopian Public Health Institute for all the

services they provided me. Lastly, I would like honestly to thank all that support me directly or

indirectly during my study period.

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 TABLE OF CONTENTS
Approval Page.....................................................................................................................................i
Declaration.........................................................................................................................................ii
Abstract.............................................................................................................................................iii
Acknowledgements...........................................................................................................................iv
Table of Contents................................................................................................................................v
List of Tables.....................................................................................................................................vi
Appendixes.......................................................................................................................................vii
List of Abbreviations.......................................................................................................................viii
1) Introduction...................................................................................................................................1
1.1) Problem Statement...................................................................................................................3
1.2) Objectives.................................................................................................................................4
1.2.1) General objective..................................................................................................................4
1.2.2) Specific objectives....................................................................................................................4
1.3) The Research Question............................................................................................................4
1.4) Significance of the study..........................................................................................................4
2) Literature Review..........................................................................................................................5
2.1) Prebiotic rich raw materials choices used in prebiotic food Formulation................................5
2.1.1) Green Banana.....................................................................................................................5
2.1.2) Carrot.................................................................................................................................6
2.1.3) Orange peel........................................................................................................................7
2.2) Processing of raw materials for formulation of prebiotic food ingredient...............................8
2.2.1) Washing.............................................................................................................................8
2.2.2) Peeling...............................................................................................................................9
2.2.3) Cutting...............................................................................................................................9

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 2.2.4) Blanching.........................................................................................................................10
2.2.5) Drying..............................................................................................................................10
2.3) Prebiotics as functional food..................................................................................................12
2.3.1) Why prebiotics?...............................................................................................................13
2.3.2) Origin of the term............................................................................................................13
2.3.3) Criteria for classifying a food ingredient as prebiotic.....................................................13
2.3.4) Sources of Prebiotics.......................................................................................................15
2.4) Mechanism of action of prebiotic..........................................................................................15
2.4.1) Modulation of the Gut Microbiota...................................................................................15
2.4.2) Immune System...............................................................................................................16
2.4.3) Anti-pathogenic activity..................................................................................................17
2.4.4) Mineral Absorption..........................................................................................................17
2.5) Benefits from prebiotics.........................................................................................................18
2.6) Isolation and characterization of prebiotics...........................................................................19
2.7) Dietary requirements..............................................................................................................21
2.8) Prebiotics in disease...............................................................................................................22
2.8.1) Prebiotics and GI disorder..................................................................................................22
2.8.2) Prebiotics and cancer..........................................................................................................22
2.8.3) Prebiotics and respiratory diseases.....................................................................................23
2.9) Prebiotics Safety.....................................................................................................................23
2.10) Effect of prebiotic in sensory acceptance of foods.................................................................24
2.11) Probiotics.................................................................................................................................24
2.11.1) Microorganisms Used as Probiotics.................................................................................25
2.11.2) Isolation and identification of probiotics..........................................................................26
2.12) Antioxidant Assessment..........................................................................................................27
2.13) Technological properties of powder........................................................................................28
2.13.1) Water absorption capacity................................................................................................29
2.13.2) Oil absorption capacity.....................................................................................................29
2.13.3) Bulk density......................................................................................................................29
3) Material and Method...................................................................................................................31
3.1) Flour processing.....................................................................................................................31
3.1.1) Green banana Peel powder processing............................................................................31
3.1.2) Preparation of Carrot Powder processing........................................................................31

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 3.1.3) Preparation of orange peel powder processing................................................................32
3.2) Composite Powder formulation.............................................................................................32
3.3) Product Formulation (soup formulation) using composite powder.......................................32
3.4) Analysis of proximate composition.......................................................................................32
3.4.1) Moisture...........................................................................................................................32
3.4.2) Ash...................................................................................................................................32
3.4.3) Fat....................................................................................................................................32
3.4.4) Protein..............................................................................................................................32
3.4.5) Total Crude fiber..............................................................................................................33
3.5) Extraction of prebiotics from composite powder...................................................................33
3.6) Bacterial Strains, Media and Substrates.................................................................................35
3.6.1) Preparation of Test Solutions and Inoculation of probiotic bacteria...............................35
3.6.2) Enumeration of Probiotic Bacteria..................................................................................35
3.7) Determination of prebiotic properties....................................................................................35
3.7.1) Resistance to acidic digestion.............................................................................................35
3.8) Determination of Technological functions.............................................................................36
3.8.1) Water holding capacity....................................................................................................36
3.8.2) Oil holding capacity.........................................................................................................37
3.8.3) Bulk Density....................................................................................................................37
3.8.4) Swelling capacity.............................................................................................................37
3.9) Determination of antioxidant in composite powder...............................................................38
3.9.1) Hydrogen peroxide scavenging assay.................................................................................38
3.9.2) Phosphomolybdenum assay................................................................................................38
3.10) Sensory evaluation of composite powder................................................................................38
3.11) Statistical Analysis.................................................................................................................39
4) Result and Discussion..................................................................................................................40
4.1) Proximate compositions analysis...........................................................................................40
4.1.1) Moisture Content.............................................................................................................40
4.1.2) Crude Protein Content.....................................................................................................41
4.1.3) Crude Fat Content............................................................................................................41
4.1.4) Ash Content.....................................................................................................................42
4.1.5) Total crude fiber..............................................................................................................42
4.2) Technological Properties........................................................................................................44

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 4.2.1) Water Holding Capacity (WHC).....................................................................................45
4.2.2) Oil Holding Capacity (OHC)...........................................................................................46
4.2.3) Bulk density.....................................................................................................................47
4.2.4) Swelling capacity.............................................................................................................47
4.3) Extraction of prebiotics from composite powder (inulin extraction by ethanol)...................49
4.4) Determination of prebiotic properties (acidic digestion resistance)......................................52
4.5) Antioxidant Assessment.........................................................................................................55
5.5.1) Phosphomolybdate assay....................................................................................................55
4.5.2) H2O2 radical scavenging activity........................................................................................55
4.6) Probiotic growth stimulation..................................................................................................57
4.7) Sensory evaluation.................................................................................................................60
4.8) Optimization...........................................................................................................................62
6) Conclusion and Recommendation................................................................................................63
6.1) Conclusion.................................................................................................................................63
6.2) Recommendation.......................................................................................................................63
Reference......................................................................................................................................64
Appendix.......................................................................................................................................72

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 LIST OF TABLES

Table 2.1: Plants with high level of inulin………………………………………………………...19

Table 2.2: Human studies with different level of prebiotic intake…………………………….......23

Table 4.1: proximate composition (%) of composite powder……………………………………..43

Table 4.2: Technological properties of composite powder………………………………………..48

Table 4.3: Content of inulin in composite powder………………………………………………...51

Table 4.4: Acidic digestion of composite powder………………………………………………....53

Table 4.5: Antioxidant assessment………………………………………………………………...55

Table 4.6: Log colony forming unit of probiotic bacteria in composite powder………………….59

Table 4.7: The Mean ± Standard Deviation Value for Sensory Evaluation……………………….61

Table 4.8: Lack of fit, adj R2 and model (prob>F) of sensory evaluation…………………………61

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 APPENDIXES

Appendix 1: Laboratory experiment figures…………………………………………………...71

Figure 1.1: Extraction of inulin………………………………………………………………...71

Figure 1.2: Growth of probiotic bacteria……………………………………………………….72

Figure 1.3: Proximate composition…………………………………………………………….73

Figure 1.4: Acidic digestion……………………………………………………………………75

Figure 1.5: Antioxidant determination ………………………………………………………...77

Figure 1.6: Sensory evaluation…………………………………………………………………79

Figure 1.7: Technological properties…………………………………………………………...81

Appendix 2: ANOVA tables for sensory evaluation……………………………………………82

Figure 2.1: ANOVA table for Aroma…………………………………………………………..82

Figure 2.2: ANOVA table for Color…………………………………………………………....83

Figure 2.3: ANOVA table for flavor…………………………………………………………...84

Figure 2.4: ANOVA table for Taste……………………………………………………………85

Figure 2.5: ANOVA table for Overall acceptability…………………………………………...85

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 LIST OF ABBREVIATIONS

AD: Atopic Dermatitis

ANOVA: Analysis of Variance
AOAC: Association of Analytical Chemistry
BD: Bulk Density
CRC: Colorectal Cancer
CVD: Cardiovascular Disease
DP: Degree of Polymerization
DV: Daily Value
GBF: Green Banana Flour
IBD: Inflammatory Bowel Disease
LAB: Lactic Acid Bacteria
LcFOS: Long Chain Fructooligosaccharide
MFGM: Milk Fat Globule Membrane
NEC: Necrotizing Enterocolitis
OHC: Oil Holding Capacity
RRF: Relative Response Factor
RS: Resistant Starch
SC: Swelling Capacity
SCFA: Short Chain Fatty Acid
ScGOS: Short Chain Galactooligosaccharide
WHC: Water Holding Capacity

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 1) Introduction
In recent years, the manufacture of functional foods incorporating prebiotic ingredients has become a
major focus in the food manufacturing industry. It's a really promising market, not only in terms of
economics, but also in terms of scientific evidence of its advantages. Consumers are becoming more
aware of the link between excellent nutrition and good health, and they are increasingly looking for
foods that give both nourishment and considerable health benefits (Burgain et al., 2011).

Prebiotics are functional foods, which are defined as a component of an edible product with a
demonstrable health effect that is not attributable to absorption in the bloodstream or the
component's sole action. The presence of the prebiotic component and the formulation in which it is
inserted alters the composition or activity of the microbial flora in the target host by modulating it,
promoting the proliferation of a select group of beneficial colon bacteria while suppressing the
proliferation of microorganisms that are harmful to health (K.M.Sreenivas et al 1998).

A prebiotic was first defined as ‘‘a non-digestible food ingredient that beneficially affects the host
by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the
colon, and thus improves host health’’. Since its introduction, the concept of prebiotics has attracted
much attention, stimulating scientific as well as industrial interest. According to this definition, only
a few compounds of the carbohydrate group, such as short and long chain β-fructans [fructo-
oligosaccharide (FOS) and inulin], lactulose, and galacto- oligosaccharide (GOS), can be classified
as prebiotics. However, many food components, especially many food oligosaccharides and
polysaccharides (including dietary fiber), have been claimed to have prebiotic activity without due
consideration to the criteria required. Not all dietary carbohydrates are prebiotics, and clear criteria
need to be established for classifying a food ingredient as a prebiotic. These criteria are 1)
resistance to gastric acidity, to hydrolysis by mammalian enzymes, and to gastrointestinal
absorption; 2) fermentation by intestinal microflora; and 3) selective stimulation of the growth
and/or activity of those intestinal bacteria that contribute to health and well-being. (Marcel
Roberfroid et al 2008).

Prebiotics are extremely beneficial to human health. Many prebiotics have been shown to have
good bile adsorption capacity in vitro. Dietary fiber's ability to bind bile salts and hence lead to
elimination through feces shows its ability to lower cholesterol levels (Lund, et al, 1989). Prebiotics
are vital in the formation of short chain fatty acids, in addition to bile salt adsorption (SCFA).

1
SCFAs such as acetate, propionate, and butyrate are key end products of anaerobic fermentation in
the large intestine in humans, and they have considerable health benefits such as anti-cancer
properties. Furthermore, SCFA are reported to meet 60–70% of the energy requirements of
colonocytes (Belenguer, et al 2008). Asparagus, sugar beet, garlic, chicory, onion, Jerusalem
artichoke, wheat, honey, banana, barley, tomato, rye, soybean, human and cow's milk, peas, beans,
and other dietary foods contain them naturally. It was recently discovered that seaweeds and
microalgae absorb it. Because they are found in low concentrations in foods, industrial
manufacturing on vast sizes is required. However, certain prebiotics are made from raw materials
such as lactose, sucrose, and starch. The majority of prebiotics classed as GOS and FOS may be
produced on an industrial scale. There is also other research on their creation that have been
published elsewhere (Marcel Roberfroid et al 2008).

The fibers found in the peel of the banana are one of the most important sources that have been
researched when attempting to increase the non-digestible fraction. Fibers, like resistant starches,
are not digested by humans due to a lack of enzymes that can break down their structure. Because
undigested fiber is commonly fermented by the colonic flora, which produces a variety of health-
promoting by-products. The main byproduct of green bananas is flour, which is one of the most
prevalent methods of preserving bananas and their masses. It has a high starch content and is
commonly utilized as a source of energy in newborn feeding. It also has excellent therapeutic
qualities, particularly for gastrointestinal infections (Martinez et al.2009). In addition to fiber and
vitamins, carrots provide minerals needed to keep healthy life. They contain calcium, iron,
magnesium, phosphorus, potassium, copper and manganese. Among other things, these minerals
help maintain bone and tooth health, proper muscle function and a healthy nervous system, and they
also help with energy metabolism, protein synthesis, fluid balance and red blood cell formation.
While carrots do not contain large quantities of minerals, eating carrots as part of a balanced diet
will help to meet daily mineral needs, (Burgain et al., 2011). Orange peel has a potential valuable
composition that can be developed into high quality products. According to Rivas et al. (2008),
orange peel composed of 16.9% soluble sugar, 9.21% cellulose, 10.5% hemicelluloses and 42.5%
pectin. Mamma et al. (2007) mentioned that orange peel is a raw material which can be direct
utilized in daily life such as animal feed and organic fertilizer. Orange peel is by products during
the processing of fruit and studies show that they are good sources of bioactive compounds. Every
year a large amount of oranges byproduct wastes are formed such as peels Manthey .A et,al 2001.
During the production of orange juice and other orange products, the orange peel accumulates in the
bulk and will produce environmental problem. Therefore, it is essential to find the applications for

2
these peels. The orange peels are rich in nutrients and contain many phytochemicals; therefore, they
can be useful in many drugs and food items.

Keeping in to consideration the above-mentioned viewpoints, the overall goal of this study was to
develop and evaluate prebiotic food ingredient, with improved dietary fiber and sensory attributes
using the green banana peel, carrot bagasse and orange peel. The developed product is expected to
help to mitigate the different health problems that happen because of inadequate consumption of
prebiotic.

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 1.1) Problem Statement
The post-harvest losses of perishable (vegetable and fruits) food crops amounted to be about 30%
(Fekadu M. et.al 2006) due to the presence of high moisture content (65–95%), insect infestation
and damage during post-harvest handling techniques (packaging, storage and transportation). The
issue of food losses is of high importance in the efforts to combat hunger, raise income and improve
food security. Ethiopia’s agricultural potential for food production is immense and over 90% of its
export earnings come from this sector. Post-harvest losses of such commodities are serious problem
particularly in Ethiopia. Between 30% and 50% of the food produced worldwide is wasted each
year. From a sustainability standpoint, adequate food waste (FW) management has been a chronic
challenge for many governments across continents. The social, environmental, and economic
effects of FW are well-known on a local and worldwide scale. Food is largely regarded as a
disposable item, with about a third of all food produced for human use being reportedly wasted
either purposefully or unintentionally. Gross mismanagement, as well as an unclear long-term
future plan for FW management (FWM), demand immediate action. Unfortunately, despite the
obvious current and future issues, an examination of the literature reveals that this topic lacks
verified records (Thyberg KL, et al, 2004) Generally, a huge amount of waste in the form of liquid
and solid is generated in fruit and vegetable processing industries, which contains many reusable
substances that have high value of economic potential. The processing industries generates high
amounts of wastes such as peels, seeds, stones, and unused flesh in the different steps. It may cause
pollution issues if not, utilized or disposed-off properly. However, a disposal of these materials
usually represents a problem that is further aggravated by legal restrictions.

Several studies have found that the chemical composition of these wastes from the preparation of
fruits and vegetables contains a variety of important nutrients. Some of these wastes are high in
critical nutrients such as carbohydrates, proteins, lipids, minerals, fibers, prebiotics, and other
nutrients. Prebiotics found in the wastes of vegetables and fruits have numerous health benefits,
including keeping your gut happy and healthy, assisting in proper nutrient absorption, lowering the
risk of cardiovascular disease, increasing immune function and reducing inflammation in the body,
maintaining a healthy weight, lowering the risk of cancer, and maintaining a healthy blood pressure.
As a result, in addition to its nutritional value, consuming a suitable amount of prebiotics is essential
for good health.
Prebiotics can be used to offer a double benefit such as an improved organoleptic quality and a
better-balanced nutritional composition (Nelson AL et.al 2002) As fiber ingredients the use of inulin
and nondigestible oligosaccharides is simple and often leads to enhance taste and texture. Specific

4
colonic bacteria, such as bifidobacteria and lactobacilli species readily ferment these particular forms
of dietary fibre and by raising their cell population with the parallel production of short-chain fatty
acids (Sghir A, et. al 1998). Both important technological characteristics and interesting nutritional
properties have been shown by Prebiotics (Huebner J, et.al 2007). Some of them are found in fruits
and vegetables and some can be processed industrially from renewable materials. In foods they affect
considerably by improving organoleptic characteristics, enhancing both taste and mouth feel. As
functional food ingredients prebiotics must be chemically stable to processing treatments of food,
such as high temperature, low pH, and Maillard reaction conditions. Therefore, if the prebiotic was
degraded to its component mono- and disaccharides or chemically altered, a prebiotic would no
longer provide selective stimulation of beneficial microorganisms and it was unavailable for bacterial
metabolism (Bohm A, et.al 2005). A study ascertained the effect on the prebiotic activity by
processing conditions of commercial prebiotics using a prebiotic activity assay

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 1.2) Objectives
1.2.1) General objective
 To develop and evaluate prebiotic food ingredient by using green banana peel, carrot
bagasse and orange peel composite powder.

1.2.2) Specific objectives

 To extract and characterize the prebiotic inulin from the formulated composite powder of
green banana peel, carrot bagasse and orange peel.
 To determine proximate composition of green banana peel, carrot bagasse and orange
peel composite powder as prebiotic food ingredient.

 To evaluate the growth of probiotics in the formulated composite powder of green banana
peel, carrot bagasse and orange peel.
 To optimize the f or mu la te d soup from green banana peel, carrot bagasse and
orange-peel composite powder based on sensory evaluation.

1.3) Significance of the study

Prebiotics are present in natural products, but they may also be added to food. The purpose of these
additions is to improve their nutritional and health value. The presence of prebiotics in the diet may
lead to numerous health benefits. Studies on colorectal carcinoma demonstrated that the disease
occurs less commonly in people who often eat vegetables and fruit. This effect is attributed mostly
to inulin and oligofructose. Among the advantages of those prebiotics, one may also mention the
reduction of the blood LDL (low-density lipoprotein) level, stimulation of the immunological
system, increased absorbability of calcium, maintenance of correct intestinal pH value, low caloric
value, and alleviation of symptoms of peptic ulcers and vaginal mycosis. The organizations,
especially food and food related organizations will work to supply the prebiotic food ingredient,
improve the selection of prebiotic food ingredient and; the researchers will use the same future
research for the literature review and for the further study in Ethiopia.

6
 2) Literature Review
2.1) Prebiotic rich raw materials choices used in prebiotic food formulation
The choice of raw materials for formulating prebiotic food ingredient is determined by different
such as nutrient density of vegetables and fruits, complementary feeding practices, socioeconomic
factors and availability of ingredients (CODEX 1991). Some examples of commonly utilized
vegetables and fruits for prebiotic food in Africa and other developing countries are onion, orange,
banana, garlic, carrot, etc. some vegetables and fruits are typically rich in prebiotic substances but
others are limiting in prebiotic i.e. some vegetables and fruits are generally high in prebiotic but
others have only low levels of prebiotic. Fruits, in general, contain a substantial amount of
micronutrients and antioxidants and their incorporation increases the nutritional quality of
functional foods. Most fruits are recognized for their vitamin C, fiber, antioxidant and mineral
contents (Nelson AL et.al 2002).
2.1.1) Nutritional significance of banana peel
Banana stands out in the green stage due to its high starch/resistant starch concentration, attracting
industrial attention for innovative product development. Furthermore, both the pulps and peels
contain a wide range of vitamins and minerals (Lima et al., 2000). Green banana mass, a material
formed by grinding fresh bananas, has been explored for food production of green banana pulp
mass (GBPM) and green banana peel mass (GBPM) since the mid-1990s (GPPM). Because of its
high starch content, it has acquired popularity in the manufacture of breads, gnocchi, pates,
mayonnaise, spaghetti, and other foods (Borges et al., 2009;). Because of its potential to restore
intestinal functions, green banana is a highly recommended diet for a variety of pathological
diseases, including constipation and diarrhea. It is advised in cases of colitis, ulcerative colitis,
gastric ulcer, uremia, nephritis, gout, cardiovascular disease, and celiac disease because it has the
capacity to increase the proliferation of good acidophilus bacteria in people. Bananas are considered
prebiotic useful foods while they are in their green stage. The most important factor for considering
green bananas as a prebiotic food is its resistant starch content, characterized by the portion of the
granule or its degradation products that are not digested or absorbed in the small intestine and are
fermented in the large intestine (Teixeira et al., 1998).

Another part of the banana that can be mentioned when one aims at increasing non-digestible fraction is
the fibers contained in the peel. Just as resistant starches, fibers are not digested by the human organism

7
 due to the absence of enzymes capable of digesting this structure. Thus, undigested fiber is fermented by
the colonic flora, which generates extremely interesting by products for human health. Flour, the main
byproduct of green banana, is one of the most common forms of preserving bananas as well as their
masses. It has high starch content and is widely used in infant feeding as a source of energy and also has
excellent medicinal properties, especially for cases of gastrointestinal infection (Martinez et al., 2009).

Some of the outcomes from the recent studies using green banana relevant to the present objective
have been summarized in table 1.

Table 1: Proximate composition (%) of green banana peel powder

Moisture 12.9

Ash 0.13

Protein 0.04

Fiber 0.84

Fat 2.08

Vitamin c (mg/100g) 0.09

Total sugar 6.00

Reducing sugar 4.00

Source: Sulaiman F. et.al 2011

2.1.1) Carrot
Carrot (Daucuscarota L) is one of the important nutritious root vegetables grown throughout the
world. It is an excellent source of phytonutrients such as phenolics, polyacetylenes and carotenoids
(Babic et al, 1993). The main physiological function of carotenoids is as precursor of vitamin A
(Nocolle et al, 2003). Carotenoids are potent antioxidants present in carrots which help to neutralize
the effect of free radicals. Reports have showed that they have inhibitory mutagenesis activity thus

8
 Contributing to decrease risk of some cancers (Dias, 2012) In recent times, consumption of carrot
and its products has gained wide acceptance as a result of its natural antioxidants properties
coupled with the anticancer activities of β-carotene in it which is a precursor of vitamin A.
Consequently, consumption of carrot and its products would be very useful in alleviating vitamin
A deficiency particularly, among children below six years and adults.
Carrots bagasse are a low-calorie food that also contains important nutritional fiber, making them a
satisfying complement to any weight-loss program. Carrots also include vitamin A and a variety of
minerals that are essential for maintaining good vision and avoiding nutritional deficits. Carrots are
more than 88 percent water by weight, making them a great high-volume, low-calorie item to add to
your weight-loss or weight-management routine. You'll receive 50 calories, 1 gram of protein, and
less than 1 gram of fat from 1 cup of carrot sticks. Carrots' low-fat content makes them a good fit
for cholesterol-lowering diets. A low-fat diet may also help men avoid prostate cancer, according to
a January 2010 report published in "The Journal of Urology." Carrot sticks provide 12 grams of
carbohydrate per cup, with 6 grams coming from natural sugars and 3.4 grams from dietary fiber.
Men require 30 to 38 grams of fiber per day, while women require 21 to 25 grams per day,
according to the Institute of Medicine. Despite the fact that this fiber is indigestible by your body, it
plays an important part un your health. Getting adequate fiber aids in correct digestion, may help
prevent colon cancer, and can help keep cholesterol levels in check. A high-fiber diet may also
benefit in weight loss and obesity prevention, according to a review of scientific studies published
in the journal "Nutrition" in June 2004. (Babic and colleagues, 1993).
Carrots include nutrients that are necessary for good health, in addition to fiber and vitamins.
Calcium, iron, magnesium, phosphorus, potassium, copper, and manganese are all present. These
minerals aid in the maintenance of bone and dental health, appropriate muscular function, and a
healthy nervous system, as well as energy metabolism, protein synthesis, fluid balance, and red
blood cell development, among other things. While carrots may not contain a lot of minerals,
including them in a well-balanced diet can help you satisfy your daily mineral requirements. (Lima
et al., 2000). Some of the outcomes from the recent studies using carrot bagasse relevant to the
present objective have been summarized in table 2.

9
Table 2: Proximate composition (DM) of carrot bagasse powder.
Moisture 10.98
Carbohydrate 6.04
Protein 9.38
Lipid 2.21
Ash 6.61
Total dietary fiber 75.76
Insoluble dietary fiber 65.77
Soluble dietary fiber 9.98
Source: Beom J, et.al 1998

2.1.2) Orange peel

The orange peel is considered as having certain vital nutrients and having certain properties which
make the gastro intestinal tract function well and it is excellent for the diabetic and heart patient as
well. Besides the nutritional aspect it is having the affordable aspect as well. A segment membrane
of citrus fruits appears to be able to prevent prostate and other cancers by acting as mediator in cell
communication, a factor known to reduce the likelihood of abnormal cell growth. Sour fruits as
lemon appear to have the greatest effect (Liu et al. 2001). The dietary fibers are the indigestible,
fibrous parts of citrus fruits are an essential part of our diet. Because of their water retaining
properties, fibers help food pass through the gut faster and therefore have a laxative effect. Fibers
add bulk to the diet and fill up, making less likely to snack on fatty foods. Therefore, we need to eat
fibers every day as part of a balanced diet (Youssef, et al 2007).

Oranges are juicy, sweet citrus fruits known for being high in vitamin C. It’s perhaps less well
known that orange peels are also rich in several nutrients, including fiber, vitamin C, and plant
compounds like polyphenols. In fact, just 1 tablespoon (6 grams) of orange peel provides 14% of
the Daily Value (DV) of vitamin C — nearly 3 times more than the inner fruit. The same serving
also packs about 4 times more fiber (Food data Centre USDA,). One test-tube study found that the
total polyphenol content and activity in orange peels was significantly higher than in the actual fruit
(Jae-Hee, et al, 2014,).
Specifically, orange peels are a good source of the polyphenols hesperidin and
polymethoxyflavones (PMFs), both of which are being studied for their potential anticancer effects
(Shafya, et al, 2018,).

10
Additionally, nearly 90% of the essential oils in orange peels are made up of limonene, a naturally-
occurring chemical that has been studied for its anti-inflammatory and anticancer properties,
including against skin cancer (Hakim, 2000, et al). Some of the outcomes from the recent studies
using orange peel relevant to the present objective have been summarized in table
Table 3: Proximate composition of orange peel powder.
Moisture 9.50
Protein 5.17
Crude fat 4.41
Total ash 2.53
Sugar 9.20
Total dietary fiber 74.14
Indigestible dietary fiber 55.47
Digestible dietary fiber 19.10
Source: Gorinstein S. et.al 2001.

Orange

Soaking/Washing

Grating

Freeze Drying

Particle Size
Reduction

Ground Orange
Peel

Packaging

Storage

Figure 2.1: Process flow chart for the manufacture of orange peel powder.

11
 2.2) Processing of raw materials for formulation of prebiotic food ingredient
2.2.1) Washing
Fruits and vegetables are frequently engaged in food-borne outbreaks because they contain a wide
variety of microorganisms. Because fruits and vegetables are typically consumed raw or with
minimum processing (as in ready-to-eat salads), microbiological safety becomes a critical
consideration to reduce consumer risk (Sagoo et al. 2003). A number of recent outbreaks have been
linked to fresh-cut fruits and vegetables, and have been linked to poor hygienic conditions.
Investigations into these outbreaks revealed that the quality of the washing water was critical. One
of the most important processing phases in the manufacturing of fresh-cut fruits and vegetables is
disinfection. This phase frequently has an impact on the ultimate product's quality, safety, and shelf
life. To improve quality, washing is used to remove dirt, select pesticides, and detach germs.
Sanitization is the process of killing contaminating microorganisms after they have been washed
away. Mechanical washing in the presence of sanitizers is used in chemical cleaning and sanitizing
processes (Artés et al, 2005). Sanitizers can minimize contaminating pathogens and lower the
growth of indigenous microbial populations on the surface of fresh-cut vegetables by 2–3 log units.

12
 2.2.2) Peeling
Peeling is an important aspect of the food processing process, and the majority of agricultural crops
must be peeled in order to be removed at the start of the process. Peeling fruits and vegetables
eliminates the inedible parts (peel, seeds, and stalk). The sensitivity to spoiling, on the other hand,
increases as physiological processes speed up and tissues are exposed to microbes. Peeling may
reduce the shelf life and quality of fresh-cut fruits and vegetables. The goals of optimum peeling
operation are (Radhakrishnaiah. Setty et al. 1993):
1. Minimizing product losses, Types of products e.g., potato products,
2. Minimizing heat ring formation e.g., apple, potato,
3. Minimizing energy and chemical usage, and
4. Minimizing the environmental pollution
Peeling operation can be grouped under following categories:
• Manual peeling (knife or blade),
• Mechanical peeling (abrasive devices, devices with drums, rollers, knifes or blades and milling
cutters),
• Chemical peeling,
• Enzymatic peeling, and
• Thermal peeling (Flame or dry heat peeling, steam or wet heat peeling, thermal blast peeling,
and vapor explosion or vacuum peeling.

2.2.3) Cutting
Non-thermal food operations for size reduction include cutting, slicing, dicing, and shredding.
Consumers' preparation time is reduced as a result of this approach. Using a knife, chopper, or
slicer, cut out inedible and discolored sections of items. Injured tissues, on the other hand, are
removed during food processing to prevent germ spread. Because sliced tissues have less
respiration and enzyme activity, they take longer to deteriorate and have a longer shelf life.
Cutting collects fluids on the cut surface, which enhances microbial load and enzyme activity
(Das et al. 2011).

13
 2.2.4) Blanching
Blanching is a unit operation that involves heating fruits or vegetables to inactivate enzymes,
change texture, preserve color, flavor, and nutritional content, and remove trapped air before
freezing, canning, or drying. In industry, the most frequent heating media for blanching are hot
water and steam, but microwave and hot gas blanching have also been investigated. Various hot
water and steam blanchers have been developed to improve product quality, yield, and processing
of products with various thermal properties and geometries. Recent improvements in equipment
design have been driven by energy conservation and waste minimization. Although blanching
appears to be a straightforward process, heat transfer to a delivered bed of product and its effects on
product qualities are extremely difficult to estimate using predictive mathematics. Although
enzymes are normally inactivated during processing, other quality factors such as color and texture
are frequently checked. Typically, the mass flow rate is fixed for a certain product, the temperature
is measured, and the heating medium flow rate is adjusted to keep the temperature at the set point.
(Bruzzese and colleagues, et.al 2009)

2.2.5) Drying
The earliest method of food preservation is drying. The sun, wind, and a smoky fire have all been
used to remove water from fruits, meats, cereals, and plants throughout history. Food dehydration is
defined as the process of eliminating water from food by circulating heated air across it, which
prevents enzymes and bacteria from growing. Dried foods are delicious, nutritious, light, easy to
prepare, store, and utilize. The amount of energy required is lower than that required to freeze or
can, and the storage space required is far smaller than that required for canning jars and freeze
containers. Drying has only a minor impact on food's nutritional value. Although vitamin A is kept
during drying, food containing it should be stored in dark places because it is light sensitive.
Vitamin A is abundant in yellow and dark green vegetables such as peppers, carrots, winter squash,
and sweet potatoes. Heat destroys vitamin C, however pre-treating meals with lemon, orange, or
pineapple juice boosts the vitamin C concentration. Dried fruits and vegetables are high in fiber,
carbs, and fat, making them a healthy meal option. (Fedorak and colleagues, et.al 2004).
2.2.5.1) Sun Drying
Fruits can be dried in the sun because of their high sugar and acid content (fig 2.2). Vegetables have
a low sugar and acid content. This raises the chances of food spoiling.

14
A minimum temperature of 86 degrees Fahrenheit is required, with higher temperatures preferable.
Foods must be dried outside for several days. Sun drying might be dangerous because to the
unpredictability of the weather. Sun drying is best when the humidity is below 60%. When the fruit
ripens, these optimal conditions are frequently unavailable. Fruits that have been sun-dried are
arranged on screens or wooden dowels. Screens must be safe to touch when they come into contact
with food. Stainless steel, teflon-coated fiberglass, and plastic screens are the best
(Radhakrishnaiah. Setty et al. 1993). Screens constructed of "hardware cloth" should be avoided.
This is a cadmium- or zinc-coated galvanized metal cloth. These substances can oxidize and leave
hazardous residues on food. Copper and aluminum screening should also be avoided. Copper
reduces vitamin C absorption and promotes oxidation. Aluminum corrodes and discolors easily.
Most timbers are suitable for building trays in an outdoor drying rack. Green wood, pine, cedar,
oak, or redwood, on the other hand, should not be used. These woods cause food to distort, discolor,
or have off-flavors. Place trays on blocks to improve air circulation around the food. Because the
ground could be wet, it's better to set up the racks or screens on a concrete driveway or, if feasible,
on a sheet of metal or tin. The sun's reflection on the metal raises the drying temperature. To keep
the fruit safe from birds and insects, cover the trays with cheesecloth. Fruits that have been exposed
to the sun must be covered or brought under cover at night. The cool night air condenses and could
add moisture back to the food, thus slowing down the drying process. (Cho, et al 2009).

Figure 2.2 Sun drying of vegetables and fruits

2.2.5.2) Oven Drying

A dehydrator is available to anyone who owns an oven. An oven can be used as a dehydrator by

15
combining the factors of heat, low humidity, and air flow (fig 2.3). An oven is perfect for drying
fruit leathers, banana chips, or preserving extra produce such as celery or mushrooms on a regular
basis. Because the oven is used for everyday cooking, it may not be adequate for storing plentiful
garden food. Because there is no built-in fan for air flow, oven drying takes longer than
dehydrators. (Some convection ovens, however, do feature a fan.) Drying food in an oven takes
nearly twice as long as it does in a dehydrator. Thus, the oven is not as efficient as a dehydrator and
uses more energy. (Gupta A, et al, 1997).

Fig 2.3 Oven drying

2.3) Prebiotics as functional food
Any fresh or processed food that claims to have a health-promoting and/or disease-preventing
feature in addition to its basic purpose of giving nutrients is referred to as functional food or
therapeutic food. Food ingredients that fit into these functional food criteria and are of interest to
the food industry are prebiotics and probiotics. Prebiotics are dietary carbohydrates that have
selective metabolism in the colon and serve to increase the number of probiotics such as lactic acid
producing bacteria, including Bifidobacteria. Probiotics are live microbial feed supplements added
to appropriate food vehicles, whereas prebiotics are dietary carbohydrates that have selective
metabolism in the colon and serve to increase the number of probiotics such as lactic acid
producing bacteria, including Bifidobacteria. Prebiotics have received more attention in recent
years for their potential health benefits, including as better resistance to invading microorganisms,
improved intestinal function, colon cancer prevention, cholesterol reducing activity, and improved
calcium and iron absorption (Bruzzese et al 2009). Since one of the most prominent trends in
today's food sector is the search for functional foods or functional food ingredients. Both prebiotics
and probiotics have great business value and biological potential. Prebiotics are fermented by the

16
microflora inhabiting the host's gastrointestinal system, selectively increases the growth and/or
activity of one or a restricted number of bacteria within the host's gastrointestinal system (Gibson,
et al, 2004), non-probiotic gut flora such as Bacteriodes sp. and Escherichia coli do not digest it
(Fedorak, et al, 2004).

Although certain biomolecules may meet the first two criteria, the third and fourth criteria are more

17
difficult to meet. This necessitates taking into account fermentable substrates in the human diet, as
well as microbiota availability and selective metabolism. It needs anaerobic sampling followed by
reliable and quantitative microbial analysis of a wide variety of microbial genera i.e. total aerobes,
total anaerobes, bacteriodes, bifidobacterium, clostridium, enterobacteria, eubacterium and
lactobacillus. (Gibson et al 2004).

There are five main criteria for the classification of food components such as prebiotics, according
to (Wang, et al, 2009). Prebiotics are not digested (or are only partially digested) in the higher
portions of the alimentary canal, according to the first criterion. As a result, they make their way to
the colon, where they are fermented selectively by possibly beneficial bacteria (a condition of the
second criterion) (Maccfarlane, et al, 2008). Increased stool mass, a mild reduction in colonic pH, a
reduction in nitrous end products and faecal enzymes, and an enhancement in the immune system
are all possible outcomes of fermentation. (Crittenden, R, et al, 2009), which is beneficial for the
host (the requirement of the third criterion). Selective stimulation of growth and/or activity of the
intestinal bacteria potentially associated with health protection and wellbeing is considered another
criterion (Gibson, et al, 2004). The classification's last criterion assumes that a prebiotic can
withstand food processing conditions while being intact, non-degraded, or chemically unmodified
and ready for bacterial metabolism in the colon (Wang, et al, 2009,). Huebner et al. (2008) used
diverse processing settings to examine six commercially available prebiotics. In varied processing
circumstances, they discovered no significant changes in the prebiotic activity of the studied
compounds. Meanwhile, Zee et al. (2012) shown that starch may be used to change the ability of
gut microbes. Prebiotics should have a well-documented structure, and components utilized in
pharmaceutical formulae, food, or feed additives should be relatively simple to get on a large scale
(Angelakis, et al, 2017). However, it should be noted that taking too many prebiotics might cause
flatulence and diarrhoea, but taking too many probiotics has no such negative effects. Prebiotics can
be taken for a long time and as a preventative measure. Furthermore, when taken correctly, they do
not produce any side effects such as diarrhoea, UV sensitivity, or hepatic damage caused by
antibiotics. Prebiotics aren't allergic, and they don't promote the spread of antibiotic resistance
genes. Of course, the effect of using prebiotics to eliminate specific bacteria may be inferior to
antibiotics, but the features listed above make them a natural antibiotic replacement (Crittenden, R,
2009, et al).

18
 2.3.1) Sources of Prebiotics
Soybeans, inulin sources (such as Jerusalem artichoke, jicama, and chicory root), uncooked oats,
unprocessed wheat, unrefined barley, and yacon are all good sources of prebiotics. Some of the
oligosaccharides found naturally in breast milk help newborns build a healthy immune system.
Lactobacilli and Bifidobacteria, which are part of the baby's protection against pathogens and a key
precursor for the immune system, govern the flora of breast-fed infants. The oligosaccharides in
breast milk, which are considered the original prebiotic, nourish these floras. While various
peptides, proteins, and lipids have been studied as possible prebiotics, non-digestible carbohydrates,
particularly non-digestible oligosaccharides, have gotten the most interest (Anandharaj, et al, 2014).

Fig 2.4 Prebiotic origin

19
 2.4) Mechanism of action of prebiotic
2.4.1) Modulation of the Gut Microbiota
Prebiotics change the composition of the gut microbiota, leading to a rise in health-promoting
organisms like bifidobacteria and lactobacilli, according to evidence from human feeding
experiments. These bacteria are normally safe because they primarily ferment carbohydrates, are
not pathogenic, and are non-toxigenic, but they do play a role in colonization resistance and
frequently exhibit immune modulatory qualities in their hosts. Some species may also convert
prebiotics like acetate and butyrate to SCFAs like acetate and butyrate, which are vital energy
sources for the host. Despite the fact that bifidobacteria do not make butyrate, they have been
proven to stimulate butyrate-producing bacteria in the gut, such as eubacteria. SCFAs are also
involved in cell proliferation and differentiation, colonic epithelial cell transport, and hepatic lipid
and carbohydrate metabolism control (Slavin, J. et.al 2013). Prebiotics have an advantage over
probiotics in that the target bacteria are already present in the host; but, if the organisms required to
promote health are not already present in the gut, for example, due to sickness, the prebiotic may
have no impact. Prebiotics have been found in certain studies to reduce the populations of specific
types of bacteria in the gut, including clostridia, bacteroides, enterococci, and enterobacteria, some
of which may be harmful to the host's health. Some of these species, particularly clostridia, are
directly toxigenic, able to break down proteins and ferment their component amino acids, producing
toxic metabolites such as indoles, phenols, ammonia, thiols, H2S, and amines, which may play a
role in colorectal cancer.The sugar composition, and degree of polymerization of the prebiotic,
together with the availability of other carbohydrates, all affect the way in which bifidobacteria (and
other saccharolytic species) are able to grow on these substances (Cho, et al, 2009).

2.4.2) Immune System

Prebiotics have been shown to have a positive impact on the immune system. It's unclear if these
are direct or indirect effects of immune modulatory bacteria or SCFA synthesis, which has been
shown to have immune modulatory qualities and can bind to SCFA G protein linked receptors on
immune cells in gut-associated lymphoid tissues (GALT). Increased mucosal immunoglobulin
synthesis, mesenteric lymph nodes, Peyer's patches, and altered cytokine generation in the spleen
and intestinal mucosa have all been linked to the addition of FOS and lactulose to the diet.
Investigations on the effects of prebiotics on the immune system require careful assessments of
the choice of markers, which will vary, and be dependent on the condition under study (Ferguson,
L. et. Al 2007)).

20
 2.4.3) Anti-pathogenic activity
In several studies, prebiotics in the diet protect the GIT from infection and inflammation by
blocking pathogenic bacteria's or their toxins' adhesion and/or invasion of the intestinal epithelium.
Glycol conjugation glycoproteins and lipids on the microvillus membrane facilitate this connection.
Prebiotics, particularly GOS, have features comparable to those found on microvillus membranes
that bind to bacterial receptors, preventing bacterial adhesion to the colonic epithelium. Prebiotics
found in human milk have been shown to have anti-adhesive qualities as well as the ability to
neutralize toxins (Roberfroid, M. et.al 2007).

2.4.4) Mineral Absorption

The capacity of prebiotics to boost calcium, magnesium, iron, and zinc absorption, as well as the
attendant augmentation of bone mineralization, is one of the most important health effects of
prebiotics on mammalian physiology. Prebiotic action on mineral absorption has been linked to a
number of pathways. Despite the fact that human studies have been restricted and modest in scale,
this could be useful in preventing osteoporosis, a prevalent and sometimes painful illness, as well as
avoiding diet-related anemia and boosting vitamin absorption to avoid malnutrition. In humans,
calcium is mostly absorbed in the small intestine, and prebiotic feeding studies have failed to show
increased calcium absorption, suggesting that prebiotics were affecting these processes in the large
intestine. Many studies have validated findings that some calcium is absorbed from the colon, and
that prebiotic metabolism increases big intestine calcium intake. This could happen through a
variety of processes. Prebiotic fermentation lowers the intraluminal pH in the large intestine,
increasing calcium solubility and bioavailability for absorption. The presence of SCFA has not been
associated to magnesium absorption, but it has been linked to the lactate pool in the gut and low pH.
Lactic acid is more acidic than SCFA, showing that the process is the direct absorption of lower pH.
(De Vrese, et.al 2008)

Short-chain fatty acids (SCFAs) such as lactic acid, butyric acid, and propionic acid are produced
when prebiotics are fermented by the gut bacteria. These items can have a variety of effects on the
human body. Propionate, for example, has an effect on T helper 2 in the airways, macrophages, and
dendritic cells in the bone marrow. SCFAs lower the pH of the colon (Bunout, D. et. Al 2002).

21
 Fig 2.5 prebiotic action of mechanism
Another prebiotics fermentation product that can boost the innate immune system's defenses against
harmful bacteria is peptididoglycan. The fermentation products are determined by the structure of
prebiotics and the bacterial composition of the gut. The effects of prebiotics on human health are
mediated through their degradation products by microorganisms. For example, butyrate influences
intestinal epithelial development (Hamer, H.M.; et al, 2008). Since SCFAs can diffuse to blood
circulation through enterocytes, prebiotics have the ability to affect not only the gastrointestinal
tract but also distant site organs (Den Besten, et al, 2013).

2.8.1) Prebiotics and GI disorder

Idiopathic inflammatory bowel disorders include Crohn's disease and ulcerative colitis. Modulation
of gut flora, such as antibiotics, prebiotics, and probiotics, is one of the most promising new
treatments (Baumgrat et al 2007). Inflammatory bowel disease (IBD) is a recurrent chronic
condition characterized by a dysregulated host microbiota interaction. Patients with IBD have been
demonstrated to have a higher chance of developing colorectal cancer. Probiotics and prebiotics
therapies, which attempt to restore gut microbiota balance and reduce intestinal inflammation, have
recently received a lot of attention. Prebiotics have been examined less extensively, but due to their
ability to boost endogenous lactobacillus and bifidobacterium, they may become an appropriate
treatment or co-treatment alternative. Prebiotics and probiotics could be a promising new treatment
option for IBD. However, in order to establish which probiotic, prebiotic, or combination thereof is
the most effective, a better knowledge of the mechanisms behind their action on the gut flora is

22
required (Geier et al 2007).

2.8.2) Prebiotics and cancer

The third most frequent type of cancer is colorectal cancer (CRC). Chemotherapy, radiotherapy,
and surgery are all linked with a high risk of consequences and are not always effective,
underscoring the need for novel treatment techniques to be developed. Prebiotics, probiotics, or a
mix of the two (symbiotic) are a revolutionary new treatment alternative. Reduced intestinal
inflammation, enhanced immune function, and anti-tumorigenic activity, binding to potential food
carcinogens such as toxins found in meat products, and a reduction in bacterial enzymes that
hydrolyze per-carcinogenic compounds such as beta-glucuronidase are all potential mechanisms for
this strategy to inhibit the development and progression of neoplasia. Probiotics and prebiotics may
be effective in the prevention of colorectal cancer, have the potential to have a significant impact on
the development, progression, and therapy of colon cancer, and may play a valuable role in colon
cancer treatment. However, there have been only a few conclusive human experiments to yet (Geier
et al 2006).

Dietary bioactive food components that interact with the immune response have a lot of promise for
lowering cancer risk. Chronic inflammation, or its downstream consequences, may be a major
mechanism that can be decreased by targeting signal transduction or utilizing antioxidant effects.
Beta-glucans are a type of macronutrient that comes in a variety of forms. There is mounting
evidence that prebiotics and probiotics can help prevent cancer, and that they can also alter the
immune system (Ferguson &Philpott et al 2007).

2.8.3) Prebiotics and respiratory diseases

Probiotics' efficacy in improving gastrointestinal functions has been extensively proven, but their
potential usefulness in the prevention of infectious respiratory disorders has not been properly
investigated. Several symbiotic preparations containing 3 to 5 strains of lactobacillus plantarums,
lactobacillus rhamnosus, and bifidobacterium lactis, lactoferrin, and prebiotics such as FOS (short-
chain fructo-oligosaccharides) or GOS were studied in a three-stage prospective, randomized,
double-blind, placebo-controlled study (galacto-oligosaccharides). The study was conducted across
three winter seasons, from 2003 to 2007, with the goal of determining the potential of various
preparations to improve digestive processes and increase the body's resistance against respiratory
infections. The severity of episodes recorded statistically significant drop in episodes. These results
demonstrated that a regular, long-term intake of various symbiotic may improve health by reducing
incidence and severity of respiratory diseases during the cold season (Pregliasco et al 2008)

23
 2.5) Benefits from prebiotics

Fig 2.6: Benefits of prebiotic (Hamer, H.M.; et al, 2008.).

2.6) Isolation and characterization of prebiotics
Because prebiotics are simple carbohydrates, determining their chemical composition and isolating
them from plant sources is difficult. Initial steps necessitate prebiotic enrichment via gradients of
aqueous ethanol extraction. Separation techniques based on TLC, HPTLC, and HPLC with
refractive index detector are required for further fractionation and characterisation.
The standard formula for inulin is GFn and Fm, where G stands for anhydroglucose and F stands
for anhydrofructose. It exists as a homologous series of oligo- and polysaccharides with varying
chain lengths in nature. Because their qualities, such as digestibility, probiotic activity and health-
promoting potential, caloric value, sweetening power, water binding capacity, and so on, differ
significantly, short chain inulins must be distinguished from their long chain analogues. To enhance
native chicory and dahlia inulin in the higher molecular weight fractions, techniques such as
ultrafiltration, selective crystallization from aqueous solution, and precipitation from solvent/water
combinations are utilized. Finally, in the presence of high concentrations of solvents such as
methanol, ethanol, and acetone, long chain inulin may be precipitated from aqueous solutions
(Frank et al 2004).1-kestose (1-kestotriose; GF2), nystose (1,1kestotetraose; GF3), and 1F-beta-
fructofuranosylnystose (1,1,1- kestopentaose; GF4) content of food and feedstuffs after extraction
with water have been reported ( De Paulo Farias D. et.al 2019). Samples were chromatographed on
an ion exchange column with alkaline sodium acetate gradient, detected with a pulsed
electrochemical detector. The method provided excellent separation, recovery and quantification of
the GFn unit of FOS.

24
The impact of extraction solvents and temperatures on monosaccharide, sucrose, and raffinose
oligosaccharide extraction yields from plant materials has been studied (Johansen et al 1996).
Toasted soybean meal, cotton seed meal, field peas, and a feed mixture were extracted in 50 percent
(v/v) or 80 percent (v/v) aqueous methanol or ethanol at 20 or 500 degrees Celsius, or at the
solvent's boiling point. The extraction temperature had a significant impact on extraction in 80
percent (v/v) alcohol, and maximum extraction was only attained at the boiling point. Water
extraction with 50% (v/v) methanol or ethanol was less heat sensitive and produced equivalent
results. Aqueous ethanol (50%, v/v) was as effective as 50% (v/v) methanol, whereas lower yield
were seen at higher alcohol strength. There was no consistent difference in the extraction yield
when comparing reflux with constant stirring and water bath with occasional for any of extraction
solvents used. Acetone has been demonstrated to be the best solvent system to increase the yield
followed by ethanol and methanol. However, for the safety reasons and food purposes, ethanol is
best choice (Frank et al 2004).
It has been claimed that a straightforward approach for isolating and purifying alpha-galactosides,
raffinose family oligosaccharides (RFOs) from legumes can be found (Gomaa E.Z. et.al 2017).
Imbibition of seeds, extraction with 50% ethanol, and precipitation of RFOs, purification of RFOs
on diatomaceous earth and charcoal, and cation-exchange chromatography were all part of the
procedure. The process produced a white, fine powder with high purity RFO preparation (90
percent for lentils and 80 percent for pea seeds, as determined by HPLC-RI analysis). 5.6 and 4.3
grams of alpha-galactosides were extracted from 100 grams of lentil and pea, respectively (Gomaa
E.Z. et.al 2017). A capillary electrophoresis method for determining raffinose family
oligosaccharides (alpha-galactosides) was devised that is quick, simple, and repeatable. Sucrose,
raffinose, starchyose, verbascose, and ajugose were determined using indirect UV detection in a
sodium tetraborate buffer with added cetyltrimethyl ammonium bromide at a moderate alkaline PH
(9.2). The detection limits were around 110g/ml, which equated to 150"320M. On the basis of
linearity investigations, the relative response factor (RRF) was derived and utilized to quantify
alpha-galactosides in lupine samples (lupinusangustifollius) (Andersen et al 2003)
In less than 30 minutes, a straightforward approach enables for the isocratic chromatographic
detection of 12 carbohydrates and sugar alcohols from a single sample. The approach worked well
on a variety of plant materials, with a focus on perennial ryegrass samples from the species
loliumperenne. The approach was simply adapted to analyze the polysaccharide inulin after acidic
hydrolysis into matching monomers without requiring any significant changes in chromatographic
settings or the use of enzyme. It has advantages for analyzing complicated combinations of

25
nonstructural carbohydrates that are frequently encountered in plant samples (Raessler et al 2008).

2.7) Dietary requirements

Due to consumption habits which changes due to age, gender and local availability, it is difficult to
estimate the need for ingesting prebiotics. For example, in American adults, the prebiotic intake is
estimated to be 10 – 15 g/day as against current recommendation for dietary fiber to be 20- 35
g/day. Similar estimates for Canadian females are 21 – 25 g/day for Canadian males is 30 – 38
g/day. Thus most adult’s dietary fiber consumptions lie well below the daily requirements.
Vegetarian populations eating wholesome cereals may not need supplementation of dietary fiber but
its content of oligosaccharides needs to be investigated (Joshi et al 1991).
Table 2.2 Human studies with different levels of prebiotic intakes
Types of prebiotics Intake g/day Effect
GOS 15 Positive effect on potentially
detrimental faecal microflora
GOS 10-40 Efficient for calcium
availability.
GOS 20 Improved calcium absorption
in post- menopausal women
by 16%.
prebiotics 0.8% Higher dosages more
0.4% bifidogenic than lower
dosages during infant trial.
FOS 10 In human bifidogenesis was
higher.
FOS 5 & 10-22 Bifidobacteria population get
elevated in 75 and 100 %
volunteers.
Milk with 2.5% FOS cultured 2.5% FOS Lowering of total cholesterol
LDL, LDL:HDL ratio
respectively by 4.4:5.4 and
5.3% in human acidophilus.
GOS + Bifidiobacterium lactis NA Protection to children against
dysentery severe illness and
reduced the need of
antibiotics.

Source: Joshi et al 1991

26
 2.8) Prebiotics Safety
Prebiotics are thought to have no serious or life-threatening negative effects. Oligosaccharides and
polysaccharides are not broken down by intestinal enzymes. They are transferred to the colon,
where the gut microbiota ferments them. As a result, prebiotics' adverse effects are largely due to
their osmotic activities. In this case, prebiotic recipients may have osmotic diarrhea, bloating,
cramps, and gas. The length of a prebiotic's chain is a key factor in the development of its adverse
effects. Prebiotics with shorter chain lengths, on the other hand, may have more negative effects.
This behavior could be explained by the fact that shorter inulin molecules are digested primarily in
the proximal colon and fermented more quickly, whereas longer chain inulin molecules are
fermented later and slower in the distal colon. The prebiotic dose, in addition to chain length, can
influence its safety profile. Prebiotics at low dosages (2.5–10 g/day) and high doses (40–50 g/day)
might produce flatulence and osmotic diarrhea, respectively. It's worth noting that prebiotics require
a daily intake of 2.5–10 g to exert their favorable effects on human health. This suggests that
prebiotics can have mild to moderate adverse effects at therapeutic levels.Most products of
prebiotics in the market have doses of 1.5–5 g per portion (Svensson, U., et al, 2014). As potential
alternatives or adjunctive therapies (symbiotic) to probiotics (Garg, et.al 2018.), prebiotics may
have similar safety concerns.

2.10) Prebiotic in sensory acceptance

Sensory analysis is a critical stage in the development of food products. Morais et al. (2014) created
a chocolate dairy dessert containing prebiotics and various high-intensity sweeteners in place of
sucrose. In comparison to the prebiotic chocolate dairy dessert containing 8% sucrose, sweeteners
had the strongest sweetening power, according to the relative sweetness analysis. Consumers want
healthier functional items with no added sugar, thus this study of sweetness in this product is
critical. Cruz et al. (2013) wanted to see how increasing prebiotic oligofructose concentrations
affected the physicochemical, rheological, and microbiological aspects product. During 28 days of
refrigerated storage, the addition of prebiotic oligofructose had no effect on the pH, proteolysis, or
viability of Streptococcus thermophilus or Lactobacillus bulgaricus (p > 0.05). Prebiotic inulin was
added to bread in another trial, and the outcomes were smaller loaves, a firmer crumb, and a darker
color. Sensory tests revealed that inulin content reduces acceptability, that yeast invertase and dry
heat destroy inulin, and that fructo-oligosaccharide/inulin fortification in bread at 5% looks feasible
(Morris & Morris, et al, 2012). The purpose of this study was to see how a prebiotic (fructo-
oligosaccharide) affected the sensory qualities and consumer acceptability of peach-flavored
drinkable yogurts. The yogurts containing the prebiotic were not significantly different from their

27
comparable controls indicating that a prebiotic can be added without impacting acceptance
(Gonzalez et al., 2011).

2.11) Probiotics
Currently, the most widely used definition of probiotics is “live microorganisms that, when
administered in adequate amounts, confer a health benefit on the host” (Hill et al.2014). While there
is no official intake level for foods, it is generally accepted that probiotics should be consumed at a
level between 107-109 CFU/g of product to ensure the health benefits (Ashraf, et al, 2011). Dietary
probiotic health benefits are achieved by the arrival of the probiotic to the gut, where they
positively impact the natural gut microbiota by increasing the growth of good bacteria and
hindering the growth of undesirable or pathogenic bacteria. Thus, potential probiotic strains must be
evaluated for specific characteristics before their use in functional foods. Orally consumed
probiotics therefore must survive transit through the digestive tract until they reach the colon, which
is the target organ. For a strain to be considered a probiotic, it should be proven safe for human
consumption. Must survive reaching the target organ by having acid and bile tolerance, and be able
to grow and colonize in the gut to promote health benefits (Gotcheva et al., 2002).

2.11.2) Isolation and identification of probiotics

Most human probiotic species are members of two genera of lactic acid bacteria (LAB):
Lactobacillus and Bifidobacterium (Fotiadis CI, et.al. 2008). In recent years, fermented milk and
yoghurt have been used as a popular vehicle for delivering probiotic bacteria in food ( Ashraf,
et.al.2011). In these products, probiotic strains of Bifidobacterium and Lactobacillus are present in
combination with other lactic acid and starter bacteria ( Quinto EJ, et.al.2014). From the microbial
aspect, the therapeutic effects or health benefits of probiotic bacteria depend on three fundamental
criteria: viability, metabolic activity, and the quantity of probiotic bacteria in products. The CODEX
standard for fermented milks (CODEX STAN 243-2003) confirms that the counts of probiotic
bacteria in the fermented milks should be at least ≥106 CFU/g or mL (CFU: colony forming units) at
the end of product shelf-life (Ashraf, R., et.al. 2011). Conventional culture-dependent methods that
identify bacterial populations to the genus level involve the isolation of pure cultures using selective
media, followed by the Gram-staining, morphological observations, analysis of carbohydrate
fermentation, detection of specific enzymes, and other biochemical tests ( Ward P, Roy D. et.al.2005).
In practice, the presence of multiple and closely related species of lactic acid bacteria in probiotic
products makes the differential or selective enumeration of probiotic and yoghurt starter bacteria
difficult, due to similarity in growth requirements and overlapping biochemical characteristics of the
species (Ashraf, et.al. 2011). Recently, alternative culture-independent (molecular) methods were

28
introduced for rapid, accurate, sensitive, and efficient identification and quantification of probiotic
bacteria (Ward P, et.al. 2005). In terms of quantification, the most widely used method is quantitative
real-time PCR (qPCR) (Ward P, et.al. 2005). However, some molecular approaches are more
applicable for taxonomic or epidemiological objectives; some of these methods are expensive and
difficult to handle in a routine manner. Wilkins-Chalgren agar containing mupirocin is claimed to be
selective for the isolation and enumeration of Bifidobacteria in fecal and probiotics samples that
contain mixed populations of lactic acid bacteria. On this selective medium, mupirocin completely
inhibits the growth of Lactobacilli, Lactococci, Leuconostocs, and Streptococci strains (Ashraf, et.al.
2011). International Organization for Standardization (ISO, 20128/IDF 192: 2006) has recommended
De Man Rogosa Sharpe (MRS) agar supplemented with clindamycin (and also ciprofloxacin in some
references) as the preferred medium for the selective isolation and enumeration of Lactobacillus
acidophilus strains in dairy probiotics products containing other lactic acid bacteria and
Bifidobacteria (Ashraf, et.al. 2011). In the present study, we assessed the selectivity of Wilkins-
Chalgren agar containing mupirocin (WCM) and De Man Rogosa Sharpe (MRS) agar containing
clindamycin/ciprofloxacin culture media in different antibiotic concentrations for probiotic
Bifidobacterium spp. and Lactobacillus spp. Afterwards, one tuf gene-based specific primer set was
designed for Bifidobacterium animalis subsp. lactis BB-12, and its specificity was determined by
PCR assays whit DNAs extracted from prevalent Bifidobacterial and Lactobacilli probiotic strains.

2.12) Antioxidant Assessment

Seidel et al (2007) reported proper intake of prebiotics and natural antioxidants incorporated into a
main food had an influence on several parameters of the immune system. An increasing amount of
data point to a combined antioxidant and immunity-modulatory effect for prebiotics, suggesting that
the underlying mechanisms might be identical. Thus, the addition of prebiotics exhibited positive
effect on the antioxidant activity. The increase of antioxidant capacity ascribed to that active
hydroxyl in molecular structure of prebiotics showed up the ability of scavenging radical (Li et al.,
2008). Early feeding of prebiotic oligosaccharides to babies benefit a lot to breastfeeding
(Arslanoglu, Moro, & Boehm, 2007).
Antioxidants are substances that can either slow or stop the oxidation processes that occur when
oxygen or reactive oxygen species are present in the environment. Polymeric products,
petrochemicals, foodstuffs, cosmetics, and medications are all stabilized with them. Antioxidants
have a role in the body's defense mechanism against diseases caused by free radicals. Enzymes like
superoxide dismutase, catalase, and glutathione peroxidase, as well as nonenzymatic substances such
uric acid, bilirubin, albumin, and metallothioneins, are examples of endogenous antioxidants. When
endogenous factors are unable to maintain strict control and total protection of the organism against

29
reactive oxygen species, exogenous antioxidants, such as nutritional supplements or pharmaceutical
medicines containing an antioxidant molecule as the active principle, are required. Vitamin E,
vitamin C, -carotene, vitamin E, flavonoids, and mineral Se are among the most important exogenous
antioxidants. Exogenous antioxidants can come from natural sources (vitamins, flavonoids,
anthocyanins, and some mineral compounds), but they can also come from synthetic sources
(butylhydroxyanisole, butylhydroxytoluene, gallates, and so on) (Litescu SC, et al. 2011).There is an
increasing interest in antioxidants, particularly in those intended to prevent the presumed deleterious
effects of free radicals in the human body, as well as the deterioration of fats and other constituents
of foodstuffs (Molyneux P. et.al .2004)

2.13) Technological properties of powder

Functional properties are the essential physicochemical properties of foods that reflect the complex
interactions between the structures, molecular conformation, compositions, and physicochemical
properties of food components, as well as the nature of the environment and the conditions under
which these are measured and associated (Suresh et al., 2009). Functional characteristics are needed
to determine whether or not new proteins, fats, carbohydrates (starch and sugars), and fiber can be
used to stimulate or replace conventional protein, fat, carbohydrates (starch and sugars), and fiber in
specific food systems, as well as to demonstrate whether or not they can be used to stimulate or
replace conventional protein, fat, carbohydrates (starch and sugars), and fiber. Functional qualities
also explain how components behave during preparation and cooking, as well as how they affect the
appearance, texture, and taste of the final product. Swelling capacity, water absorption capacity, oil
absorption capacity, Emulsion activity, Emulsion stability, Foam capacity, Foam stability,
Gelatinization, Bulk density, Dextrinization, Preserving, Denaturation, Coagulation, Gluten
formation, Jelling, Shortening, Plasticity, Flakiness, Moisture retention, Aeration, and Sensory
attributes are just a few of the functional properties. The structure, quality, texture, nutritional
content, acceptability, and (or) appearance of a food product all contribute to its functional property.
The organoleptic, physical, and/or chemical qualities of a food are frequently used to determine its
functional properties. Solubility, water retention, foaming ability, elasticity, absorptive capacity for
fat and foreign particles, emulsification, hydration (water binding), viscosity, cohesion, and
stickiness are all examples of functional qualities of foods and flour (Suresh, et al, 2013). The
components of the food material, particularly carbohydrates, proteins, fats and oils, moisture, fiber,
ash, and other ingredients or food additives added to the food (flour), such as sugar alcohols
(Awuchi, et al, 2017), as well as the structures of these components, influence the functional
properties of foods and flours. Patulin, a mycotoxin found in grains, may impair the functional
qualities of flours (Awuchi et al., 2019). Each ingredient in a food serves a specific purpose, such as

30
thickening food mixtures and forming the bulk of foods; sugars add flavor and aid in food browning;
eggs can act as an emulsifier, thickener, and foaming agent; and fats and oils play important roles in
food aeration, emulsions, and shortening, among other things. Food quality is highly dependent on
functional qualities. Gelatinization, browning, dextrinization, gelation, and other processes are
mostly caused by starch. Foaming, browning, emulsification, coagulation, denaturation, and other
processes are all caused by protein. Fat is majorly responsible for emulsification, aeration,
shortening, among others. Although two or more constituents may have the same influence, and one
may also have a minor influence on a functional property.

2.13.1) Water absorption capacity

The amount of water (moisture) taken up by food/flour to achieve the desired consistency and make
a quality food product is known as water absorption capacity (WAC). It's the amount of water that
should be added to a dough before it becomes too sticky to work with. Water absorption that is too
low or too high might have a negative impact on food quality. The weight of the food/flour is
frequently used to determine water absorption. For example, 60 percent water absorption indicates
that 60 pounds of water are required to hydrate 100 pounds of flour.

2.13.2) Oil absorption capacity

Oil absorption capacity (OAC), also known as water absorption, is the ability of proteins to bind fat
to their non-polar side chains. Oil absorption capacity is a crucial functional attribute that helps to
improve mouth feel while preserving the flavor of food products (Iwe et al., 2016). In diets with a
high protein content, the rate of oil absorption is extremely high. Protein's ability to bind oil and
water in meals is determined by intrinsic characteristics like protein structure, amino acid content,
and surface polarity or hydrophobicity (Suresh, et al, 2013). Flours' propensity to bond with oil
makes them beneficial in food applications where optimal oil absorption is needed, allowing them to
have potential functional applications in meals like pastries and sausage. The oil absorption capacity
also makes the flour suitable in facilitating enhancement in flavor and mouth feel when used in food
preparation (Suresh, et al, 2013). Due to these properties, the flours with good OAC are used as
functional ingredient in foods such as sausages, whipped toppings, angel and sponge cakes, chiffon
dessert etc.

2.13.3) Bulk density

The mass of several particles of any material divided by the entire volume they occupy is defined as
bulk density, also known as volumetric density or apparent density. Flours, powders, tiny particles,
granules, and other split solids of foods have this functional feature (or food ingredients). Particle
volume, internal pore volume, and inter-particle void volume all contribute to the total volume

31
(Buckman, et al, 1960). Bulk density is a feature of food materials that changes depending on how
they are handled; it is not an inherent property of food materials. When flour is placed into a
cylinder, for example, it has a given bulk density; when the cylinder is disturbed, the flour particles
move and often settle closer together, resulting in a higher bulk density. For this reason, the bulk
density of flours is often reported both as "tapped" density and "poured" (or "freely settled") density,
where the tapped density refers to the flours’ bulk density after a specified compaction process, often
involving vibration of the container (The Powder process, 2018). In some instances, the cylinder is
continuously tapped until there is no noticeable change in volume of the flour or food powder.
The swelling index (SI), also known as the swelling capacity (SC), is the volume in milliliter taken
up by the swelling of one gram (1 g) of food under specified conditions. Its determination is based on
the addition of water or a swelling agent to each individual food material as specified in the test
protocol (whole, pulverized, or cut). The swelling capacity reflects the degree of associative forces in
the starch granules as well as the starch's ability to absorb water and swell. In some food products,
such as bread goods, swelling capacity (index) is used as a quality indicator. It is a sign of non-
covalent bonding between the molecules of starch granules, as well as one of the -amylose
components. (Iwe et al., 2016). The particle size, species variety, and manner of processing or unit
activities all influence the swelling capacity (index) of flours (Suresh, et al, 2013).

32
 3) Material and Method
The raw materials are obtained from different geographical of Ethiopia. Green banana was from
Gofa zone (Sawilla Town), carrot was from Gedeo Zone (Dilla area) and orange was from Sidama
province (Wondogenet). When bananas (Musa acuminate) are to be sent to distant they are best
harvested in hard mature but unripe green state, which reduces the risk of deterioration during
transport. Bananas have a very short post-harvest life at ambient conditions. This is four to ten days.
The assessment of the readiness of orange fruit for harvest presents some problems because: citrus
fruits do not ripen further after harvest. To reach their full flavour and sweetness they must be left
on the tree to ripen. Orange (Citrus sinensis) fruits can be held up to three weeks under ambient
conditions, depending upon the temperature and moisture content of the air. In dry air they may lose
moisture and shrink after a few days. Damaged fruit may become infected and decay quickly after
harvest. Oranges: at 0 to 90C, 90-95% Relative Humidity, for 3 to 8 weeks. Harvested fruit is taken
in the harvesting container directly to the packing facility, where it is emptied into field containers.
At this point the fruit should be protected from exposure to sunlight and rain while awaiting
packing or movement to the packing house. Carrots (Daucus carota) are ready to harvest about 90
days or more after seeding, but continue to grow and enlarge them. Size is the best maturity index.
Harvest when the roots are of good size, but still tender. If carrots are left too long in the soil or
allowed to over-mature, the roots become tough, woody and start cracking. At optimal storage
conditions mature carrots can be stored at 00 C and 95-98% Relative Humidity up to 6 to 9 months.

All the chemicals and reagents which were important used for this study were in analytical grade
and were purchased from Addis Ababa.The chemicals and reagents used in this study were, HCl
(India), H2SO4(China), Hexane(China), MRS Agar, Hydrogen per Oxide, Sodium
Hydroxide(India), Mono and Di basic Sodium Phosphate(India),Citric Acid(China) Ammonium
Molybdate(China) and Ethanol(Brazil). The laboratory equipment which was available at Addis
Ababa Science and Technology University in food process engineering laboratory were utilized.

The apparatus and equipment used in this research were petri dish, incubator(Thermo-3900, India),
desiccator, water bath(Polys-400, China), centrifuge (Cole-pormer FS-3500, Canada), crucibles,
muffle furnace(FB-1415, Canada), conical flask, UV spectrophotometer (Shimadzu-1800, China),
measuring cylinder, balance, centrifuge tube, sample holder, heating plate(Cera-1200, China) and
pipette. All other materials that are not available in laboratory were purchased from local market in
Addis Ababa. The probiotic bacteria propagations were carried out in Biotechnology laboratory,
AASTU. The rest experiment were conducted in laboratory of Chemical Engineering.

33
Metagenics Ultra Flora Synergy probiotic tablets (from Ethiopian public health institute) was used in
this research for growth experiment using composite powder as carbon source (Lactobacillus
acidophilus NCFM and Bifidobacterium lactis Bi-07)

3.1) Flour processing

3.1.1) Green banana Peel powder processing
Bananas were washed and peeled to remove from the flesh and peels were cut into 2-3 cm pieces.
Peel slices were dipped in a 0.5 percent (w/v) citric acid solution for 10 minutes to prevent
enzymatic browning, rinsed, and sun dried for one week. The dried peels were ground in a
laboratory grinder and sieved at 177µm to obtain a fine powder of uniform size, which was then
stored in an airtight container at 4 °C until further usage (Ovando-Martinez, et.al 2009)

3.1.2) Preparation of Carrot Powder processing

The method described by Marvin (2009) was used in the preparation of carrot bagasse powder. The
carrot bagasse were washed with distilled water, slice into 56mm thickness; the slice carrots
bagasse was blanched for 3 minutes in hot water containing sodium metabisulphite to prevent
browning and discoloration. The sulphited carrots bagasse were immediately cooled by exposing to
air and sun dried for five days. The dry carrot bagasse was grounded to fine powder and sieved with
a 177μm sieve and packaged in black polythene bag for further uses.

34
 3.1.3) Preparation of orange peel powder processing
The sweet orange peel flour was prepared, as the method described by Okpala and Akpu (1998). The
ripe sweet orange fruits were hand-peeled and sliced into thin slices (1 cm thick) with a sharp
stainless steel knife after being cleaned with distilled water. Sun dried to constant weight for six
days, processed the peel slices were milled into powder and sieved through a 177 μm sieve. The sweet
orange peel flour was packaged in high-density polyethylene bag before use.

3.2) Composite Powder formulation

Green banana (A), carrot (B), and orange peel (C) composite flours treatments were selected the ratios based
on Box- Benken Experimental design recommendation of amount of flour required for each treatment. The
raw materials (green banana peel, carrot bagasse, and orange peel) and their levels were chosen based on the
literature and preliminary experiments.
Table 3.1: Three Factors and Three levels Box-Benken Experimental design
Factors Unit Minimum level Medium level Maximum level

Green banana grams -1 (20) 0 (25) +1 (30)

peel
powder
Carrot bagasse grams -1 (30) 0 (35) +1 (40)
powder
Orange peel grams -1 (15) 0 (20) +1 (25)
powder
Table 3.2: Box- Benken experimental design for three factors

Level of factor
Std Run
Coded Actual (grams)

Green Carrot Orange peel Green Carrot Orange peel

banana peel bagasse(B) powder (C) banana bagasse(B) powder(C)
powder(A) peel
powder(A)
8 1 0 +1 +1 25 40 25

11 2 -1 0 +1 20 35 25
17 3 0 0 0 25 35 20

15 4 0 0 0 25 35 20
10 5 +1 0 -1 30 35 15
5 6 0 -1 -1 25 30 15

35
 3 7 +1 -1 0 30 30 20
9 8 -1 0 -1 20 35 15

1 9 -1 -1 0 20 30 20
12 10 +1 0 +1 30 35 25

6 11 0 +1 -1 25 40 15

7 12 0 -1 +1 25 30 25
18 13 0 0 0 25 35 20
4 14 0 +1 0 25 40 20
13 15 0 0 0 25 35 20
16 16 0 0 0 25 35 20
14 17 0 0 0 25 35 20
2 18 -1 +1 0 20 40 20

3.3) Product Formulation (soup formulation) using composite powder.

The ingredients were weighed and mixed in different proportions to form eighteen different
formulations. Formulation contained water as required, sugar 3g, 5g of composite powder from
each treatment and spice (onion powder, garlic powder, coriander, pepper) were added in same
proportion1.5g each to all the formulations and heated on hot plate for five minutes. Vegetable soup
was taken as a control sample.

3.4) Analysis of proximate composition

3.4.1) Moisture
The moisture content was measured by oven drying at 105 0C overnight as outlined by AOAC
(1995). 2g of composite powder for the treatment was weighed and subjected to moisture test.

3.4.2) Ash
Ash content was measured using muffle furnace, which was kept at 550 0C for 24 hours. The ash
content of the composite powder was determined. AOAC (1995) procedure was followed for ash
determination.

3.4.3) Fat
Fat content of composite powder was measured by Soxhlet ether extraction process method. This
analysis was carried out by AOAC (1995) outlined procedures.

36
 3.4.4) Protein
Kjeldhal method was adopted to analyze the protein content in the composite powder and
procedures were followed as outlined in Official Methods of Analysis of AOAC International
(1995).

3.4.5) Total Crude fiber

The AOAC International (1995) method was used to calculate crude fiber. A 600 mL long beaker
was filled with about 2 g of flour samples. 200 mL of hot 1.25 percent H 2SO4 was added to the
mixture. Beaker was placed on digestion apparatus with warmed plates, cooked for 30 minutes, then
gravity filtered through Whiteman paper. With distilled water, the beaker was rinsed. The residue
from the paper back was transferred to a beaker holding 200 mL of hot 1.25 percent NaOH. The
previous steps were repeated. The residue-covered paper was placed in a crucible, dried overnight at
100°C, cooled in a desiccator, and reweighed (weight A). The samples were put in furnace at 600˚C
for 6 hrs. cooled in a desiccator and reweighed (weight B). The loss in weight during incineration
represents the weight of crude fiber.

3.4.6) Determining the total sugar

The total sugar was determined using the method specified by AOAC International (1995).. The
sample was dissolved in 100 mL distilled water, and 10 mL strong HCl was added to the solution,
which was then warmed in a water bath for 8 minutes before being neutralized with NaOH. The
solution was diluted further with distilled water and filtered up to 200 mL. Following that, 25 mL of
mixed Fehling's solution was pipetted into a conical flask, followed by 15 mL of the burette solution.
When the solution started to boil, three drops of methylene blue were added. Further quantities of the
solution were added from the burette (1 ml at a time) at 10 seconds interval to the boiling liquid until
the indicator was completely decolorized. The titre values obtained corresponded to the sugar content
reported in mg/100 ml.

3.4.7) Determining reducing sugar

A.O.A.C. described methods for determining reducing sugars. In 50 ml of distilled water, about 10 g
of the material was dissolved. The solution was filtered after being produced up to 100 mL with
distilled water. Pipette about 25 ml of the mixed Fehling solution into a conical flask, followed by 15
ml of the solution from the burette. The solution was heated, and three drops of methylene blue were
added when it reached a boil. A few additional drops of the solution were added to the boiling liquid
1 ml at a time from the burette until the indicator was completely decolored.The titre values obtained
corresponded to the sugar content reported in mg/100 ml.

3.5) Extraction of prebiotics from composite powder

37
Inulin was extracted with ethanol by adding 100 mL of 40 percent ethanol, boiling at 60°C for 15
minutes, and centrifuging at 1400 g/15 minutes. The extracts filtered with a Buchner funnel, then
concentrated up to 35 ml in a roto vapor. This extract is kept at a temperature of -8°C.
Proteins, lipids, and other components were removed from the extracts before they were purified.
To avoid hydrolysis, sodium phosphate was added to the extract to bring it to a pH of 7.6, and then
proteins and lipid impurities were precipitated by heating the mixtures at 70 0C for 15 minutes. The
purified extracts were concentrated on a heating plate at 90°C with continual stirring speed until the
saturation concentration of extract (250Bx at 90°C) was reached. The volume of the concentrated
extract was measured once the saturation concentration was reached. Ethanol was added to a 60
percent (v/v) concentration and allowed to sit at room temperature for 12 hours. Inulin precipitates
at these circumstances, whereas other chemicals like saponins and phenolic compounds remain in
solution and can be removed. The precipitates were filtered and dried. The filtered particles (inulin
powder) were then weighed (Fuentes Campos. et.al 2013).
The size of the fructans was determined using the pure extracts as well. The addition of ethanol to
all treatments yielded eighteen fructans treatments with various average molecular weights;
consequently, the alcohol concentration ranges that correspond to each treatment fraction. The
intrinsic viscosity of inulin (η) solutions was measured by viscometer. The molecular weight (
M ⍵ ) and degree of polymerization (DP) of inulin powder from the different fructans for all
a

treatment were calculated using Equation (1) and Equation (2), respectively:

ƞ=K × M ⍵a ................................. (1)

Where: K and α, are the empirical constants of Mark-Hounwink, 6.76 × 10-3 and 0.71,
respectively.
a
M ⍵ = 162󠇝×DPINU ………………… (2)
Where: M ⍵ ais the molecular weight of inulin and 162 is the molecular weight of a fructose or
glucose residue that makes up the inulin molecule.

38
 3.6) Bacterial Strains, Media and Substrates
3.6.1) Preparation of Test Solutions and Inoculation of probiotic bacteria
The probiotic was from Etiopian public health institute and transport to the laboratory immediately
at room temperature. Do not refrigerate. Refrigeration inhibits the viability of certain organisms. Do
not transport through the pneumatic tube. Store in dark at room temperature. Growing probiotic
bacteria in MRS broth supplemented with 0.05 percent L-cystein for 48 hours at 37°C under
anaerobic (anaerobe culture jar) circumstances activated in the incubator. For each of the selected
treatment samples, a solution (2.5 mL) containing 5 mg/mL of composite powder was added to
MRS broth (10 mL). Using a vortex mixer, the tubes were fully mixed before being sterilized at
121°C for 15 minutes. The petri dishes were inoculated with a loop-full of bacteria after chilling
(Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07) and they were incubated
anaerobically in anaerobic incubator under 37 ºC for 24 hours.

3.6.2) Enumeration of Probiotic Bacteria

Using the spread plate approach, Serial dilutions are used to calculate the concentration of
probiotic. As it would usually be impossible to actually count the number of microorganisms in a
sample, the sample is diluted and plated to get a reasonable number of colonies to count. Since the
dilution factor is known, the number of microorganisms per ml in the original sample can be
calculated. Count the number of colonies on each plate. If there are too many colonies on the plate,
the colonies can run together and become indistinguishable as individual colonies. In this case the
plate is called confluent or Too Numerous To Count (TNTC). The countable plate has between 30
and 300 colonies. More than 300 colonies would be difficult to count, and less than 30 colonies is
too small a sample size to present an accurate representation of the original sample. As stated
above, the number of colonies is the number of Colony Forming Units which represents the number
of microorganisms per ml. To find out the number of CFU/ ml in the original sample, the number of
colony forming units on the countable plate is multiplied by 1/dilution factor (Herigstad, et al.,
2013).

3.7) Determination of prebiotic properties

3.7.1) Resistance to acidic digestion
The ability of composite powder to resist digestive processes such as gastric acidity, breakdown by
mammalian enzymes, and gastrointestinal absorption is termed as non-digestibility. The degree of
hydrolysis of composite powder is measured when they are exposed to prepared Hydrochloric acid
buffer.
The analysis was based on the findings of a study conducted by (Wichienchot, et.al 2006).

39
Hydrochloric acid buffer contains (in g/L): NaCl, 8; KCl, 0.2; Na2HPO42H2O, 8.25; NaHPO4, 14.35,
CaCl22H2O, 0.1; MgCl26H2O, 0.18. 5 M HCl was used to change the pH of this buffer from 1 to 5.
This is a buffer (5 mL at each pH) was added to sample solution (1% w/v, 5 mL) and incubated in a
water bath (37 ± 1 °C) for 4 h. Percentage of hydrolysis of sample was calculated based on reducing
sugar released and total sugar content of the sample as below:

Where: reducing sugar released is the difference between its final and initial content.

3.8) Determination of Technological functions

3.8.1) Water holding capacity
The Water Holding Capacity (WHC) was calculated using Traynham TL et .al 2007.
In pre-weighed 30 mL plastic centrifuge tubes, 2.5g composite powder was weighed. 10 mL
distilled water is added to each sample and well mixed with it. For 30 minutes, samples were kept at
room temperature (22°C). For 30 minutes, the mixture was centrifuged at 1200 g (3709 rpm). The
supernatant was carefully decanted shortly after centrifugation, and the sample's new mass was
recorded. The following formula was used to determine WHC (g water / g powder):

3.8.2) Oil holding capacity

With minor adjustments, Chakraborty's Oil Holding Capacity (OHC) was determined. In a pre-
weighed 30 mL plastic centrifuge tube, 2g composite powder was weighed. 20 mL refined vegetable
oil (sunflower oil, density = 0.89877 g. ml-1) was added to each sample and well mixed with the
sample using a Vortex mixer set to the highest speed; the samples were then allowed to stand at room
temperature (22°C) for 30 minutes. The sample-oil mixture was centrifuged for 30 minutes at 1200 g
(3709 rpm), the supernatant was properly decanted, and the sample's new mass was recorded (Megha
AV, et. al 1986). The OHC (grams of oil per gram of dry powder) is calculated as follows:

Where md and moiled are the mass of dry material and the mass of sample including held oil,
respectively.

3.8.3) Bulk Density

40
Composite powder of 100g, M, weighed with 0.1 percent accuracy, into a dry graduated 250-mL
cylinder (readable to 2mL) without compacting. If necessary, level the powder carefully without
compacting it, then convert the unsettled apparent volume (V0) to the nearest graded unit. Using the
formula m/V0, calculate the bulk density in g/mL (Iwe M. et.al 2016).

41
 3.8.4) Swelling capacity
Okaka and Potter (1977) revised a method for determining swelling capacity. The sample was filled
to the 10 ml mark in a 100ml graduated cylinder. To make a total volume of 50 ml, distilled water
was added. By inverting the cylinder, the top of the graded cylinder was tightly covered and
blended. After 2 minutes, the suspension was inverted again and permitted to stand for another 8
minutes, and the volume occupied by the sample was measured was taken after the 8th min.

3.9) Determination of antioxidant in composite powder

3.9.1) Hydrogen peroxide scavenging assay
The composite powder's ability to scavenge hydrogen peroxide (H2O2) was tested using the
method of Ruch et al 2017. A 2g sample was placed into the tubes, which were then filled to 0.4
mL with 50 mM phosphate buffer (pH 7.4) before adding 0.6 mL of H2O2 solution (2 mM). The
reaction mixture was vortexed, and its absorbance was measured at 230 nm after 10 minutes of
reaction time. A positive control was employed, which was ascorbic acid. The following calculation
was used to calculate the composite powder's ability to scavenge H2O2:

Where: A0= Absorbance of control, A1= Absorbance of sample

3.9.2) Phosphomolybdenum assay
The method of Prieto et al. 1998 was used to carry out the phosphomolybdenum test. 1 mL of
reagent solution was used to treat a sample of 2g composite powder (0.6 M sulfuric acid, 28 mM
sodium phosphate and 4 mM ammonium molybdate). The tubes were incubated in a water bath at
95°C for 90 minutes. At 765 nm, the absorbance of the samples was measured after they had been
cooled to room temperature.
A positive control was employed, which was ascorbic acid. The following equation was used to
calculate antioxidant capacity:

42
 3.10) Sensory evaluation of composite powder
The soup was tasted by ten (10) expert panelists from Addis Ababa Science and Technology
University's food process engineering department. Allow participants to read and sign a consent
form acknowledging their willingness to take part in the sensory evaluation. On a 9-point Hedonic
scale (Meilgaard et al, 1999), ask the panelists to rate the composite on its aroma, flavor, color,
sweetness, and overall acceptability (Response Variables): 9=like extremely, 8=like very much, 7
=like moderately, 6 = like slightly, 5 = neither like nor dislike, 4=dislike slightly, 3=moderately, 2=
dislike very much, and 1= dislike extremely. After sensory evaluation the optimum composite
powder condition was obtained by optimization.

3.11) Statistical Analysis

Using the Statistical Package for Social Sciences, perform a one-way analysis of variance
(ANOVA) on the data (Mintab version 18.1, International Business Machines, New York, U.S.A.).
Using Mintab software, version 18.1 and ANOVA, analyze the sensory data (Mintab Institute, Inc.,
Cary, North Carolina, U.S.A.). Tukey's HSD is used to separate means or compare significant
differences, and the statistical significance is denoted as p < 0.05.

43
 4 Result and Discussion
4.1Proximate composition
4.1.1 Moisture Content
The moisture content of the composite powder formulation for sample 1 (25g of green banana peel
powder, 40g of carrot bagasse powder, and 25g of orange peel powder) was higher of 14.5% and
followed by composite powder formulation for sample 14 of 13.3% (25g of green banana peel
powder, 40g of carrot bagasse powder, and 20g of orange peel powder) , and composite powder
from sample 6 (25g of green banana peel powder, 40g of carrot bagasse powder, and 20g of orange
peel powder) was lower value of 3.1%. (Table 4.1). Chakkaravarthi et al 1993 investigated the
grinding characteristics of carrots and found that the moisture content of the dried grits had a
significant impact on the grinding energy, which increased as moisture content increased from 10%
to 15%, decreased as moisture content rose to 18%, and increased again as moisture content rose to
higher values. As a result, the moisture level of 18% was recommended for grinding operations
since it required the least amount of grinding energy. The result of samples 1, 10, 11, 14 and 18
were above 10% which have an opportunity of spoilage by mold than the other samples and had the
values of 14.5%, 10.5%, 12.0%, 13.3% and 12.5% respectively. The rest samples had the values of
below 10% moisture content. The results of this investigation were closely related to those reported
by Oppong et al 2015 for banana peel flours' Proximate Composition and Some Functional
Properties. Most composite powder tests had moisture content below 10%, which is suitable for
long-term flour storage. The moisture content of samples 3 and 13, 4 and 16 were 9.0% and 8.5%
respectively. The type of food, the variety of food, and the storage circumstances all influence the
moisture content of a food (Oppong et al., 2015). The samples 6, 9 and 12 had more low moisture
content of 3.1%, 3.4% and 4.5% respectively. Flour's low moisture level improves its storage
stability by inhibiting mold growth and minimizing biochemical reactions (Singh et al., 2005).
Consequently, the low moisture content of sample 6 (25g of green banana peel powder, 30g of
carrot bagasse powder and 15g of orange peel powder) composite powder will extend the shelf life
of the final product made from them.

4.1.2 Crude Protein Content

The crude protein content for the composite powder were listed in Table 4.1. Protein content was
higher in sample 1 (25g of green banana peel powder, 40g of carrot bagasse powder, and 25g of
orange peel powder) with the value of 7% than in the other composite powder samples. Protein
quality determination aids in the prediction of dietary protein nutritional quality.

44
The composite powder samples' crude protein concentration ranged from 0.5% to 7.0%. The protein
content of samples 1, 11, 14 and 18 were 7.0%, 5.5%, 6.5% and 6.2% respectively with maximum
carrot bagasse powder level of 40g. This is because the inclusion of a large amount of carrot
bagasse in the formulations enhanced the protein content significantly. According to the literature,
carrot bagasse has a higher protein content than orange peel and green banana peel. This rise was
expected since some root plants contain more protein than fruits, resulting in synergistic protein
complement effects (Yetunde et al., 2009).the protein content of samples 2, 3, 4, 10, 13, 15 and 16
were 4.5%, 3.4%, 3.0%, 5.0%, 3.7%, 3.2% and 3.5% respectively with medium carrot bagasse
powder level of 35g. As shown in Table 4.1, the lower percentage was 0.5 percent for 25g of green
banana peel powder, 30g of carrot bagasse powder, and 15g of orange peel powder blend in sample
6. The value produced by quantifying nitrogen in a sample using the Kjeldahl method, in which
nitrogen compounds in the sample are degraded by sulfuric acid to ammonia, sodium hydroxide is
added, steam distillation is carried out under alkaline conditions, and distilled ammonia is absorbed
in acid and measured by titration and multiplying the result by the factor. These work showed that
the increase in protein content was the direct result of the appreciably higher protein content of
carrot bagasse powder. The protein content of samples 6, 7, 9 and 12 were 0.5%, 2.0%, 2.7% and
1.0% respectively with minimum carrot bagasse powder level of 30g.

4.1.3 Crude Fat Content

Table 4.1 lists the fat content values in the composite powder. sample 1 (25g of green banana peel
powder, 40g of carrot bagasse powder, and 25g of orange peel powder) had a high fat level of 10.0
percent, but sample 6 (25g of green banana peel powder, 30g of carrot bagasse powder, and 15g of
orange peel powder) had a low fat content of 1.5 percent.

The fat level of the composite powder samples ranged from 1.5 to 10.0 percent, which is a low fat
content. This could be because fruits, roots, and tubers store energy as starch rather than lipids,
according to scientists (Iwe et al., 2016). Because all fats and fat-containing foods contain some
unsaturated fatty acids, they are possibly prone to oxidative rancidity (Reebe et al., 2000), the fat
content of samples, 5, 6, 8 and 11 were 2.0%, 1.5%, 2.4% and 2.5% which were lower than the
others , this is due to low level of orange peel powder in the formulation, the low fat levels may be
useful in ensuring extended shelf life and stability for the products (Reebe et al., 2000). As shown
in Table 4.1, composite powder samples 3, 4, 7, 13, 15 and 16 were 4.8%,5.5%,3.2%, 4.1%, 4.5%
and 4.0% respectively with medium orange peel powder level of 20g. The results were lower than
those published, ranging from 1.64 percent to 12.79 percent reported by (Iwe et al. 2016) for the
Proximate, functional and pasting properties of African yam bean and brown cowpea seeds

45
composite flour. Humans require a lot of energy, so foods with a lot of fat contribute a lot to it. In
this investigation, the fat content of samples 1, 2, 10 and 12 were 10.0%, 8.3%, 9.4% and
8.0%respectively, which were a higher fat content than the other composite powders. Flours with a
high fat content are also good flavor enhancers and can help improve the palatability of the dish
they're used in (Aiyesanmi, et al, 1996). Foods' flavors are enhanced by fats. When feeds are held
for a long time, a phenomena occurs in which moisture remains constant while ether extracts
gradually diminish. This is because unsaturated fatty acids contained in feeds are oxidatively
polymerized absorbing oxygen in the air and becomes insoluble in ether.

4.1.4 Ash Content

The mineral content of meals is determined by total ash. According to the USDA Nutrient database,
the ash level of fresh fruits and vegetables ranged from 0.3 to 1.6 percent. In Table 4.1, the ash
content of the composite powder was stated.

As shown in Table 4.1, sample 10 composite powder had the highest ash content of 18.2 percent for
30g of green banana peel powder, 35g of carrot bagasse powder, and 25g of orange peel powder,
and run 9 composite powder had the lowest ash content of 20g of green banana peel powder, 30g of
carrot bagasse powder, and 20g of orange peel powder. Sample 5 with 30g of green banana peel
powder, 35g of carrot bagasse powder, and 15g of orange peel powder composite powder had the
second highest ash concentration of 17.1 percent. The ash content readings were substantially
greater than Oppong et al (2015) reported range of 1.00 to 3.00 percent. The amount of ash in a
food is a measure of its mineral content. As a result, ash content of samples 1, 5, 7, 10, 11 and 14
were 14.8%, 17.1%, 15.5%, 18.2%, 13.0% and 14.3% respectively. The ash content of these
samples may be a more essential supplier of minerals than other composite powders. The ash
content of 7.1%, 8.5%, 6.5%, 6.0% and 7.6% with sample 2, 6, 8, 9 and 18 respectively were low as
compared to other samples. The amount of minerals in a specific food sample is indicated by ash
content, which can also be used as a quality measure for contamination (Kavitha, et al, 2014). The
ash content of food indicates the total amount of mineral elements present in the food. After organic
elements (fats, proteins, and carbohydrate) and moisture have been removed by
oxidation/incineration (Iwe et al., 2016), ash reflects the inorganic constituents' composition. It is
basically the mineral content of food sample. Minerals are essential nutrients which serve a variety
of essential metabolic functions and among the parts of molecules such as adenosine triphosphate
(ATP), haemoglobin, and deoxyribonucleic acid (DNA) (Iwe et al., 2016).

4.1.5 Total crude fiber

46
From sample 1 to 18, crude fiber increased from 69.6 percent to 84.2 percent, with sample 11
having the lowest value of 69.6 percent and sample 10 having the highest value of 84.2 percent.
Increases in green banana peel powder and orange peel powder increased the crude fiber content of
the composite powder. The samples 5 (30g of green banana powder and 15g of orange peel), 7 (30g
of green banana peel powder and 20g of orange peel) and 10 (30g of green banana peel powder and
25g of orange peel powde) had crude fiber content of 83.4%, 82.8% and 84.2% respectively, which
were higher than the other samples. This could be because green bananas and oranges are high in
fiber, according to Gidamis et al. The crude fiber, which is most likely derived from the peels of
green bananas and oranges, makes up a variable fraction of dietary fiber and mostly consists of
lignin, cellulose, and hemicelluloses (Mannay, et al, 2005). The crude fiber content of samples 2, 6,
8, 9 and 11 were 74.6%, 71.2%, 73.7%, 72.3% and 69.6% respectively. The composite product's
higher fiber and lower carbohydrate content have various health benefits, including assisting in
colon digestion and reducing constipation, which is commonly associated with products (Jideani, et
al., 2011). Dietary fiber is now widely acknowledged to play a substantial role in the prevention of
a variety of diseases, including cardiovascular disease, diverticulosis, constipation, irritable colon,
cancer, and diabetes, according to well-documented studies (Slavin, et al., 2011). The composite
powder's crude fiber content was within the FAO/WHO recommended limit for dietary fiber and
other non-absorbable carbohydrates dry matter (FAO/WHO, 1994).

Table 4.1: Proximate composition (%) of composite powder sample

samples Moisture Ash Crude Fat Protein Crude fiber
1 14.5 14.8 10.0 7.0 82.0
2 7.3 7.1 8.3 4.5 74.6
3 9.0 11.5 4.8 3.4 79.5
4 8.5 11.0 5.5 3.0 79.0
5 10.0 17.1 2.0 1.6 83.4
6 3.1 8.5 1.5 0.5 71.2
7 5.6 15.5 3.2 2.0 82.8
8 6.5 6.5 2.4 1.6 73.7
9 3.4 6.0 2.5 2.7 72.3
10 10.5 18.2 9.4 5.0 84.2
11 12.0 13.0 2.5 5.5 69.6
12 4.5 10.2 8.0 1.0 77.5
13 9.0 11.5 4.1 3.7 78.3
14 13.3 14.3 7.0 6.5 81.2
15 8.0 11.0 4.5 3.2 79.1
16 8.5 11.5 4.0 3.5 79.5
17 9.0 11.0 5.5 3.0 79.0
18 12.5 7.6 6.5 6.2 75.8
4.2 Functional Properties of composite powder

47
Various functional properties of composite flours were investigated using standard methodologies in
this study (Table 4.2). Functional properties or characteristics are intrinsic physico-chemical
properties that reflect the complex interaction between the composition, structure, confirmation, and
physicochemical properties of protein and other food components, as well as the nature of the
environment in which these are associated and measured, as reported by (Kinsella. et al 1976).
The next sections address the impact of varying flour incorporation proportions on the functional
qualities of composite flours. Food functional qualities are used to determine food attributes
throughout processing, manufacture, consumption, and storage, according to Dehnad, et al (2016). It
also regulates food quality parameters like nutritional, sensory, organoleptic, and physicochemical
qualities.

4.2.1 Water Holding Capacity (WHC)

According to Aydin, et al (2015), increasing WHC in flour can change the viscosity and texture of
food, boosting bulking, thickening, and gelling effects. The WHC of all composite powder samples
rose with increasing powder ratios, ranging from 7.02 g/g to 11.04 g/g dry samples. With 20g of
green banana peel, 40g of carrot bagasse, and 20g of orange peel (11.04g/g dry sample), Run 18
composite powder had the highest WHC value. However, with 25g green banana peel, 30g carrot
bagasse, and 15g orange peel (7.02 g/g dry sample), composite powder run 6 had the lowest WHC
value (7.02 g/g dry sample). WHC may be linked to the physical state of starch, dietary fiber, and
protein (Waliszewsld et al, 2003) in the flour. Amylase has the ability to effectively bind water
molecules, resulting in a greater WHC, according to Rodriguez-Ambriz et al, (2008). The high WHC
found in carrot bagasse and orange peel could be attributable to the dietary fibers and protein, while
resistant starch was high in green banana peel (Emaga et al., 2007). The increase in WHC in
composite powder samples was partly due to resistant starch, solution properties of dietary fiber like
hemicelluloses and pectin polysaccharides (Zhang et al., 2005), and to a lesser extent, gelatinization
of starch in the flour, which absorbs water into starch granules with concomitant swelling (Zhang et
al., 2005). (Rodriguze-Ambrize et al., 2008).

Table 4.2 shows the water absorption capacity of composite flours. The composite powder run 18
had the greatest WHC (11.04g/g) in this research. The results imply that increasing the amount of
green banana peel powder in the sample with high grams of carrot bagasse affected the amount of
water held. This could be attributed to high dietary fiber in carrot bagasse and resistant starch in
green banana peel, which impeded water absorption, as evidenced by decreasing WHC values as
proportions increased. A similar observation was made by another person (Kaushal et al. 2012).
Lower WHC in some flours may be related to reduce availability of polar amino acids in flours,

48
according to (Kuntz et al. 1971). Increased amylose leaching and solubility, as well as loss of starch
crystalline structure, could explain the increase in WHC of blends after they've been combined.
WHC elevation has traditionally been linked to increased amylose leaching and solubility, as well as
loss of starch crystalline structure. Polysaccharides and other hydrophilic elements may be present in
the powder with a high water retention capacity. Proteins can interact with water in foods because
they are both hydrophilic and hydrophobic. The good WHC of composite powder run 18 may prove
useful in products where good viscosity is required such soups and gravies. The observed variation in
different flours may be due to different protein concentration, their degree of interaction with water
and conformational characteristics (Butt et al, 2010).
The results revealed that the amount of water held was altered by the formulation of blends
containing green banana peel, carrot bagasse, and orange peel. The rise in amylose leaching and
solubility, as well as the loss of starch crystalline structure, are responsible for the significant
increase in water holding capacity of powder. The composite powder's high water holding capacity
suggests that it could be employed in the production of sausage, dough, and pastry items. (Butt and
colleagues, 2010). Mcwatters et al (2003) reported that lower water absorption is due to less
availability of polar amino acids in powder.

4.2.2 Oil Holding Capacity (OHC)

The OHC of composite powder samples increased with the addition of fiber-rich carrot bagasse and
orange peel powder, ranging from 1.52g/g to 4.35g/g dry samples. The hydrophilic nature of
starches found in flour (Rodriguez, Ambriz et al., 2008) is high in green flour (Zhang et al., 2005)
and low in ripe flour (Rodriguez, Ambriz et al., 2008). Ingredients with a high OHC can be used to
improve the texture and viscosity of food formulations, as well as to stabilize foods with a high fat
content.

It is obvious that as the amount of fiber and starch-rich carrot bagasse and orange peel powder rose,
the OHC of composite powders increased. As a result, variations in the presence of non-polar side
chains, which may bind the hydrocarbon side chain of the oil among the powders, could be a
plausible reason for the increase in the OHC of composite powder after integration of each other.
Kaushal et al. (2012) discovered similar results. However, the flours studied in this study could be
advantageous in food structure interactions, such as taste retention, palatability improvement, and
shelf life extension, especially in bread or meat items where fat retention is needed (Aremu et al.
2007). Protein, which is made up of both hydrophilic and hydrophobic components, is the most
important chemical component affecting OHC. Side chains of non-polar amino acids can generate
hydrophobic interactions with lipid hydrocarbon chains (Jitngarmkusol et al. 2008).

49
Kaushal et al. (2012) discovered similar reports, the ability of flour protein to physically bind fat
by capillary attraction is potentially useful in structural interactions in food, particularly in flavor
retention, palatability improvement, and shelf life extension of bakery or meat products, doughnuts,
baked goods, pancakes, and soup mixes where fat holding is desired (Aremu et al 2007).

4.2.3 Bulk density

The bulk density (g/ml) of powder refers to the density measured without any compression. Powder
bulk densities ranged from 0.77 to 0.95 g/ml. sample 1 composite powder (0.95 g/ml) had the highest
bulk density, followed by run 10 (0.93 g/ml), sample 14 (0.92 g/ml), and run 6 composite powder
(0.77 g/ml). The current study discovered that bulk density is dependent on the amount of flours. The
bulk desity of samples 1(90g of composite powder), 14(85g of composite powder) and 18(80g of
composite powder) were 0.95g/ml,, 0.92g/ml and 0.91g/ml respectively. When the proportions of
composite powder were increased, the bulk density of composite powder increased. Powders with a
high bulk density are suitable for use in food preparations. As a result, the current study reveals that
the composite powder with the highest bulk density is suitable for use as a thickener in food products
and in food preparation since it helps to minimize paste thickness, which is an essential factor in
convalescent and child feeding. Low bulk density, on the other hand, would be advantageous in the
formulation of supplementary foods (Akapata et. al 1999). With the addition of powders to each
other, the bulk density of composite flours grew dramatically. Similar findings have been reported by
others (Eltayeb et al. 2011).

The bulk density of food materials is significant in terms of packing benefits (Lawn 2010). High bulk
density is an useful physical property when measuring the mixing quality of a particulate matter
since it indicates the proportional volume of packaging material required (Basma., 2003). The
density of processed items determines the amount and strength of packing material used, as well as
the texture and mouth feel of the product (Fola, et al, 2011). It is clear that increase in the proportion
ratio of powder increased the bulk density of composite powder.

4.2.4 Swelling capacity

Different flours have swelling capacities ranging from 18.20 to 23.42 ml. Table 4.2 shows that the
lowest swelling capacity was found in sample 9 (18.20 ml), while the highest was found in sample 5.
(23.42 ml). The ability of flours to swell is determined by particle size, variety, and processing
methods or unit operations. According to the literature, parboiled material flour has a higher swelling
capacity than raw material flour. The degree of inclusion ratio of green banana peel, carrot bagasse,
and orange peel powders increased the swelling capacity of composite powders. The ability of starch

50
to absorb immobile water and swell is measured by its swelling capacity (Ikegwu et al, 2009). The swelling
capacity of flours varies depending on particle size, variety, and processing processes. Because green banana
peel flour is a rich source of starch, it is clear that the level of green banana peel flour had a significant impact
on the swelling capacity of composite flours. High swelling capacity has also been mentioned as a criterion
for a high-quality product (Niba et al, 2001).

Table 4.2: Technological properties of composite powder

samples OHC(g/g) WHC(g/g) BD(g/ml) SC(ml)

1 1.60 11.03 0.95 22.16
2 2.76 10.22 0.89 18.21
3 2.95 9.27 0.86 21.74
4 2.98 9.26 0.85 21.73
5 3.37 8.07 0.81 23.42
6 3.75 7.02 0.77 21.09
7 4.35 7.08 0.80 23.31
8 2.79 8.03 0.82 19.45
9 3.40 7.06 0.78 18.20
10 3.34 10.70 0.93 23.38
11 1.66 11.01 0.90 22.95
12 3.42 8.01 0.79 20.69
13 2.97 9.27 0.86 21.74
14 1.64 11.02 0.92 22.54
15 2.96 9.25 0.87 21.73
16 2.97 9.27 0.86 21.75
17 2.98 9.26 0.84 21.73
18 1.52 11.04 0.91 20.33

51
 Figure 4.2: Bar graph for technological properties of composite powder.
4.3Extraction of prebiotics from composite powder (inulin extraction by
ethanol)
Inulin, a non-digestible carbohydrate, is a fructan that is not only found as a storage carbohydrate in many
plants, but has also been part of man's diet for ages. Inulin's ability to replace fat is extraordinary. It forms
a particle gel network when thoroughly mixed with water or another aqueous liquid, resulting in a white
creamy structure with a short spreadable texture that may easily be included into dishes to replace fat by
up to 100%. (Franck A. et. al 1997). Inulin and oligofructose are two functional food components that
have a unique combination of nutritional and technical capabilities. Inulin-derived or related products have
found widespread use in the food industry, owing to their ease of incorporation with other ingredients and
ability to improve organoleptic qualities. Modified inulin has recently been discovered to be useful as a
chelating agent, a detergent co-builder, and a pharmaceutical carrier for drug administration. Inulin is sold
as a powder, with a hue that ranges from white to grey depending on the level of purification. It was
discovered that when drying in an oven between 80 and 90 °C, some inulin is hydrolyzed, resulting in
greater fructose content, based on the composition of fresh, oven dried, and sun-dried root and fruit plants.
However, sun dried roots and fruits had almost the same fructan content as in fresh. Sun-dried
roots and fruits can, therefore, be used for extraction and isolation of inulin (Abou-Arab et al.,
2011).

Inulin can be precipitated from the fruit and root extract with ethanol, according to Gupta et al. Inulin

52
precipitation from a composite powder of sun-dried green banana peel, carrot bagasse, and orange peel
with ethanol 80 percent at 90 °C after 15 minutes revealed that the low composition of run 8 (20g of
green banana peel, 35g of carrot bagasse, and 15g of orange peel) did not result in increased inulin
precipitation (1.69g) Table. As a result, after 15 minutes at 900°C, a high concentration of inulin
(2.344g) can be precipitated in run 10 (30g of green banana peel, 35g of carrot bagasse, and 25g of
orange peel). Also, from this data the supernatant obtained after precipitation of run 10
composite powder extract with 80% ethanol is a rich source of fructo-oligosaccharides because
if there was high amount of inulin then the same is true with fructo-oligosaccharides as per
literature.
Inulin is a carbohydrate that is composed of both oligomers and polymers. Green banana peel, carrot
bagasse, and orange-peel powder have the optimum integration ratios of 30g, 35g, and 25g,
respectively, according to this research. Gupta et al. investigated the effect of varying ethanol
concentrations on inulin precipitation. Inulin and oligosaccharides fractions were successfully
isolated from fruits and roots by extracting furcto-oligosaccharides using 80 percent ethanol,
according to the study. The fructan precipitation from water/ethanol extract of grains has been
reported by David P. Livingston. By using the ethanol extraction in this study obtained fructan DP
and Mw ranged from 22.1 – 41.8 and 3572.4 – 6772.4 respectively. Inulin from composite powder of
green banana peel, carrot bagasse and orange peel which produced from this study was presented on
table 2

Table 4.3 shows the molecular weight (MW) of powder inulin derived from composite powder. By
fractionating the extract with ethanol, the molecular weight distribution was found. When ethanol is
added to the combination, the solubility of fructans in aqueous media becomes imbalanced. This
solubility is also influenced by the inulin molecule's molecular weight. When ethanol is added to a
fructan solution, a whitish solid precipitates. The approach was used to precipitate fructans of
various molecular weights using varied volumes of ethanol.

Molecular mass the inherent viscosity of extracted inulin ranged from 2.24 to 3.52, according to the
results of the viscosity measurement. The Mark–Houwink expression was used to compute the
molecular weight and degree of polymerization of extracted inulin, indicating that the extracted
inulin from each treatment is higher molecular weight oligosaccharides since the degree of
polymerization was larger than 10. This confirms that the inulin isolated has a high molecular
weight.

The number of monomer units in an inulin polymer strand is known as inulin DP. The DP of inulin

53
is affected by a variety of factors, including plant sources, climate and growth circumstances,
harvest ripeness, and storage duration after harvest (Chi et al., 2011). Inulin DP was higher in
composite powder from run 10 (57) than in any other treatment. The inulin DP content of newly
extracted roots and fruits is higher than that of roots and fruits that have been preserved for four
weeks (Hevi, et al., 2017). During the artichoke storage period, the average inulin DP level
decreased, which was caused by depolymerization of high molecular weight carbohydrate
molecules (Chi et al., 2011). The amount of inulin DP in extracted inulin powder varies with
molecular weight in this study. Polymerization was high in inulin powder with a high molecular
weight. The inulin DP fraction in inulin powder was greater than 10, indicating that the fructans
were of high molecular weight. Endo-inulinase activity hydrolyzes inulin to create
fructooligosaccharides (FOS), whereas exo-inulinase activity hydrolyzes inulin to form fructose
monomers. By severing the -2.1 bond in succession, exoinulinase can create fructose. By randomly
cutting and hydrolyzing the internal bonds in inulin, endo-inulinase can create FOS (Magunwidjaja
et al., 2014). FOS (lower inulin DP) and fructose were produced after the inulin was degraded. The
inulin DP percentage of extracted inulin powder was low in run 1 (25g green banana peel, 30g
carrot bagasse, and 25g orange peel) at 22.1, but high in run 10 (30g green banana peel, 35g carrot
bagasse, and 25g orange peel) at 41.8. The function of inulin is affected by DP (Shoaib et al.,
2016). Low-DP inulin is ideal for alcoholic fermentation and FOS synthesis. Because it is low in
calories, FOS is often used as a sweetener alternative for sucrose in items such as cakes, bread,
candies, milk products, and some drinks.

Long-chain inulin fractions are slightly soluble and viscous, making them suitable for use as fat
substitutes in low-fat dairy products (Guggisberg et al. 2009). When sheared in milk or water, the
long chain inulin can also form microcrystals. As a result, inulin is commonly used to replace sugar
and fat in baked goods, ice creams, spreads, fillings, confections, and salad dressings. Although
inulin is water soluble, its solubility is completely dependent on temperature (i.e. »6% at 100°C and
35% at 900°C). It can be dispersed in water, but because of its hydroscopic nature, it can form
clumps, which can be avoided to a degree by mixing it with sugar or starch.This probably is due to
presence of high-DP crystalline fractions of inulin that do not dissolve readily (Bot et al. 2004).
Poor solubility especially for higher molecular weight fractions tends to increase at higher
temperatures (Glibowski 2010)

54
Figure 4.2: Extracted and purified inulin from composite powder.

Table 4.3: Content of inulin in composite powder

Ethanol volume for precipitation to determine DP and Mw

Samples Inulin Ethanol Mw (g/mol) DP

content (ml)
1 1.92 15 3572.4 22.1
2 1.78 20 3833.8 23.7
3 1.83 25 3974.3 24.5
4 1.84 30 4340.0 26.8
5 2.06 35 4599.9 28.4
6 1.80 40 4981.1 30.7
7 2.21 45 5804.1 35.8
8 1.69 50 6772.4 41.8
9 1.71 55 6050.9 37.4
10 2.34 60 5716.7 35.3
11 1.81 65 5751.6 35.5

55
 12 1.89 70 5891.9 36.4

13 1.84 75 6282.8 38.8

14 1.87 80 6626.2 40.9
15 1.85 85 5629.7 34.8
16 1.83 90 4748.0 29.3
17 1.84 95 5200.3 32.1
18 1.75 100 5839.2 36.0

Figure 4.3: Bar graph for extraction of inulin from composite powder.
4.4Determination of prebiotic properties (acidic digestion resistance)
A prebiotic, according to Gibson and Roberfroid (1995), is "a nondigestible food element that
enhances host health by selectively encouraging the growth and/or activity of one or a small number
of bacteria in the colon." To exert these benefits, prebiotics must be able to resist digestive processes
before reaching the colon (Gibson et al., 2004). In the stomach, digestion takes place in a very acidic
environment (pH 1-3). As a result, the composite powder was submitted to acidic hydrolyses using
HCl buffer to assess the quantities of indigestible polysaccharides in the initial evaluation of the
prebiotic qualities of the composite powder. Short- and long-chain oligosaccharides, as well as non-
starch polysaccharides including pectin, cellulose, hemicellulose, and xylans, may be found in crude
plants' "prebiotics" (Macfarlane et al., 1992).The non-digestibility of oligosaccharides has been a

56
focus of prebiotic development (Wang, Y. et al 2009) to ensure that they reach the colon and, ideally,
linger throughout the large intestine, resulting in distal effects (Gibson, G.R et.al 2004). As a result,
rather of introducing external species to the host, they can successfully activate specific indigenous
bacteria present in the gut (Musatto, S.T et.al 2007).
When compared to FOS, composite powder was found to be more resistant to simulated human
gastric juice in this study. Surprisingly, even at the lowest pH tested, the non-digestibility of
composite powder was shown to be high (pH 1). In comparison to FOS, which spans from 1.45 to
2.00 percent, it revealed minimal changes in the degree of hydrolysis within the incubation time (0 to
4 h) at pH 1 values examined (0.31 to 0.55 percent). According to the research, composite powder
was less vulnerable to gastric juice regardless of incubation period, whereas hydrolysis in the case of
FOS was shown to rise over time. Food, on the other hand, is typically held in the human stomach
for roughly 2 hours (Wang, Y. et. al 2009). The findings showed that composite powder can be
considered a viable prebiotic because it possesses prebiotic features such as non-digestibility. It was
comparable to other oligosaccharides investigated, such as kojioligosaccharides, which are 100
percent resistant to artificial stomach acid (Nakada, T et al 2003), and exhibited better resistance than
Gluconobacter oxydans gluco-oligosaccharide. When compared to FOS, a commercial and well-
known prebiotic, composite powder was found to have greater non-digestibility. In comparison to
FOS, composite powder can safely reach the colon without being severely degraded by the human
gastric juice. Because it satisfies the first feature of prebiotics, composite powder has a good
probability of being investigated further as a potential prebiotic (Aida, F.M.N.A. et.al 2009).
Essentially, the degree of digestibility indicates the compound's vulnerability to gastric juice,
allowing the remainder of the molecule to enter the colon intact and feed the probiotic bacteria. More
than 90% of a commercial prebiotic called galacto-oligosaccharide (GOS) was reported to enter the
colon (Beards, E. et.al 2010). As a result, non-digestibility of dietary components has been
recognized as one of the conditions for any prebiotic impact. Green banana peel, carrot bagasse, and
orange peel composite powder were hydrolyzed with simulated human gastric juice and found to be
resistant to the juice. At pH 1 of artificial gastric juice, the percentage of hydrolysis was minimal. At
a pH of 1, the degree of hydrolysis was 0.31 percent, 0.36 percent, 0.45 percent, 0.43 percent, 0.53
percent, and so on. In run 6 (25g of green banana peel, 30g of carrot bagasse, and 15g of orange peel)
of incubation at pH 1, the maximum hydrolysis (0.55 percent) occurred after 4 hours. In comparison
to the reference prebiotic (inulin), which showed maximum hydrolysis of 55.00 percent, 23.22
percent, 35.75 percent, and 5.96 percent at varied PHs, composite powder showed high acid
resistance. Food is often held in the human stomach, where gastric juice (pH 1–4) is produced within
2 hours; hence, when composite powders are consumed, it is predicted that a large portion of them

57
will reach the intestine. This finding is comparable to that of kojio oligosaccharide, which was
resistant to fake stomach acid 100 percent of the time (Nakada, et.al, 2003). In the acidic
environment of the human stomach, Glucono bacteroxydans produced gluco-oligosaccharide that
was 98.4 percent resistant (Wichienchot, et.al 2006). In the human digestive tract, Dextran has been
shown to be highly resistant to acidic environments (Debnam, et.al 1998).

Figure 4.3: Acidic digestion of composite powder.

Table 4.4: Acidic digestion of composite powder
samples Total sugar Final reducing Initial reducing Hydrolysis (%)
sugar (mg/100ml) sugar (mg/100ml)
1 1.34 0.34 0.27 31
2 1.31 0.29 0.50 36
3 1.40 0.54 0.21 45
4 1.38 0.50 0.22 43

58
 5 1.48 0.69 0.18 53
6 1.50 0.73 0.18 55
7 1.46 0.65 0.19 51
8 1.49 0.71 0.17 54
9 1.43 0.60 0.20 48
10 1.28 0.34 0.27 33
11 1.47 0.66 0.19 52
12 1.32 0.40 0.25 37
13 1.39 0.52 0.21 44
14 1.34 0.55 0.24 39
15 1.38 0.51 0.22 43
16 1.39 0.52 0.21 44
17 1.38 0.51 0.22 43
18 1.35 0.45 0.23 40
4.5Antioxidant Assessment
5.5.1) Phosphomolybdate assay
The reduction of phosphomolybdate ion in the presence of an antioxidant results in the creation of a
green phosphate/MoV complex, which is spectrophotometrically quantified. Table 4.5 indicates the
antioxidant capacity of composite powder in the following order: run 1 > run 10 > run 2 > run 12 >
run 14 > etc. The composite powder containing 25g of green banana peel, 40g of carrot bagasse, and
25g of orange peel had a high antioxidant capacity of 81.4 percent, followed by the composite
powder containing 30g of green banana peel, 35g of carrot bagasse, and 25g of orange peel with a
value of 30.4%. (80.8 percent). However, ascorbic acid, a well-known antioxidant utilized as a
positive control, had significantly higher antioxidant activity than composite powder.

All treatments showed varying degrees of activity in the phosphomolybdenum assay, which is a
quantitative approach for evaluating antioxidant capacity (Arabshahi-Delouee, et al, 2007). As the
concentration of orange peel powder was increased, the antioxidant activity of all composite powders
increased. However, it was discovered that run 1 of the composite powder has a large total
antioxidant capacity of 81.4 percent at a higher concentration (90g), followed by run 10 of the
composite powder (80g). The antioxidant activity of the composite powder from run 6 was the
lowest, at 55.2 percent. At concentrations of 70 to 90g, all studies revealed antioxidant action in a
dose-dependent manner. Strong antioxidant activity of run 1 statistically approximate to ascorbic
acid indicates strong antioxidants in this treatment and these could be attributable to the presence of
phenolic compounds.

The ability to decrease de Mo (VI) to Mo (V) and the ensuing information of green phosphate/Mo
(V) complex with maximum absorption at 695 nm were used to assess the total antioxidant potential
of composite powder at varied concentrations. The reaction mixture's enhanced absorbance showed

59
an increase in overall antioxidant capacity. All of the treatments in this assay had good total
antioxidant capacity, which was concentration-dependent. The overall antioxidant capabilities of
eighteen treatments ranged from 55.2 to 81.4 percent in total. It was found that all treatments
exhibited potent activities in a concentration dependent manner.

4.5.2) H2O2 radical scavenging activity

H2O2 is found in low concentrations in the air, water, human body, plants, microorganisms, food, and
beverages in our surroundings. It enters the human body by inhalation or contact with the skin. In the
body, H2O2 decomposes quickly into oxygen and water, forming hydroxyl radicals (OH•), which
can cause lipid peroxidation and damage to cell membranes and DNA. The ability of composite
powder to scavenge H2O2 radicals was ranked as follows: run1 > run 14 > run 11 > run18 > run 10
and so on. The composite powder demonstrated good H2O2 -scavenging action in the hydrogen
peroxide scavenging assay (Table 4.5). With increasing concentration, especially carrot bagasse, the
percentage scavenging activity improved. However, the scavenging activities of ascorbic acid, used
as positive controls for comparison, were relatively more evident than those of the composite powder
treatment.

H2O2 is rapidly degraded in the body into oxygen and water, which can form hydroxyl radicals
(•OH), which can cause lipid peroxidation and DNA damage (R, Cheng S. et.al 1989). In order to
evaluate if samples had the same pattern of activity as OH radical reduction ability, the ability of
composite powder treatment to scavenge hydrogen peroxide was also determined. The antioxidant
power of the plant is demonstrated by the run 1 of 25g of green banana peel powder, 40g of carrot
bagasse powder, and 25g of orange peel powder quenching hydrogen peroxide radicals. Against
hydrogen peroxide, Run 1 showed to be an effective concentration ratio (91.4 percent).

The scavenging effect of different composite powder treatment on hydrogen peroxide was
concentration-dependent (70 – 90g) as shown in table 4.5. Run1 displayed strong H2O2 scavenging
activity (91.4%) whereas that of the standard, ascorbic acid exhibited 96.7%.

Table 4.5: Antioxidant assessment

Samples Phosphomolybdate assay (total antioxidant Hydrogen peroxide scavenging assay

capacity (%) (%)
1 81.4 91.4
2 79.5 80.3
3 66.1 64.9
4 70.6 60.0
5 57.0 81.4
6 55.2 73.2
60
 7 63.7 77.4
8 56.3 79.7
9 61.4 70.7
10 80.8 85.7
11 58.2 89.3
12 78.0 74.5
13 67.7 0.52
14 75.5 0.55
15 71.8 0.51
16 69.3 0.52
17 68.2 0.51
18 73.2 0.45
4.6Probiotic growth stimulation
In MRS with composite powder, the probiotics (Lactobacillus acidophilus NCFM and
Bifidobacterium lactis Bi-07) were cultured. Table 4.6 summarizes the findings, which reveal that
cultivations were not inhibited in terms of probiotic growth when assessed using the spread plate
approach. Using log CFU/ML of 7.14 and 8.12, respectively, the cultivation with run 10 (30g of
green banana peel powder, 35g of carrot bagasse powder, and 25g of orange peel powder) showed
the maximum growth of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07. This
result was analogous to that of (Nowak et al. 2017), which looked at the ability of polysaccharides
extracted from 53 mushroom species using ethanol and distilled water to boost the metabolism of
helpful microbes like Lactobacillus strains. Mushroom polysaccharides boosted probiotic growth
more effectively than commercial prebiotics like FOS and inulin in that study.

Bifidobacterium lactis Bi-07 colonies were found to be white, glistering, and medium in size. The
bacteria were gram-positive and rod-shaped. After 48 hours, the stimulation of Bifidobacterium
lactis Bi-07 growth by composite powder ranged from log (CFU/mL) = 6.02 in the presence of run 8
(20g of green banana peel powder, 20g of carrot bagasse powder, and 15g of orange peel powder) to
log (CFU/mL) = 7.14 in the presence of run 10 (30g of green banana peel powder, 35g of carrot
bagasse powder, and 25g of orange peel powder), as shown in In comparison to inulin log (CFU/mL)
= 7.75 in the literature, eighteen treatments of composite powder increased the development of
Bifidobacterium lactis Bi-07good. Run 10 (30g of green banana peel powder, 35g of carrot bagasse
powder, and 25g of orange peel powder) was the treatment that significantly increased the
development of Bifidobacterium lactis Bi-07. The net growth of Bifidobacterium lactis Bi-07 was
boosted by the majority of these treatments in a way that was equivalent to inulin. As an example,
7.12 log CFU/mL of Bifidobacterium lactis Bi-07 were found in Run 1 (25g of green banana peel
powder, 40g of carrot bagasse powder, and 25g of orange peel powder). 7.10 log CFU/mL of
Bifidobacterium lactis Bi-07 were found in Run 12 (25g of green banana powder, 30g of carrot

61
bagasse powder, and 25g of orange peel powder, run 2 (20g of green banana peel powder, 35g of
carrot bagasse powder and 25g of orange peel powder) had 7.09 log CFU/mL of Bifidobacterium
lactis Bi-07 and Run 7 (30g of green banana peel powder, 30g of carrot bagasse powder and 20g of
orange peel powder) had 7.08 log CFU/mL of Bifidobacterium lactis Bi-07. The observed growth
stimulatory effects of these composite powders of green banana peel, carrot bagasse and orange peel
could be due to the presence of high crude fibre, resistant starch or inulin. The greater crude fibre
content of Run 10 corresponds to its higher stimulatory effects on Bifidobacterium lactis Bi-07
growth. Different regions of plants showed stronger growth stimulus on probiotics, according to
Kassim et al. (2014). In the current investigation, the peeling of selected raw materials from various
geographical regions also stimulated the growth of Bifidobacterium lactis Bi-07. Gourd family foods
have strong prebiotic activity on Bifidobacterium lactis Bi-07, according to Sreenivas and Lele
(2013). Similarly, Run 10 increased Bifidobacterium lactis Bi-07 growth more than any other
treatment in the current study.

Lactobacillus acidophilus NCFM colonies were white, glistering, and medium in size. Gram-positive
rods were the bacteria. As indicated in Table 4, log colony counts of Lactobacillus acidophilus
NCFM ranged from 6.98 for Run 4 (25g green banana peel powder, 35g carrot bagasse powder, and
20g orange peel powder) to 8.12 for Run 10 (30g green banana peel powder, 35g carrot bagasse
powder, and 25g orange peel powder). Eighteen composite powders made from green banana peel,
carrot bagasse, and orange peel stimulated Lactobacillus acidophilus NCFM growth in a way that
was similar to inulin, log (CFU/mL) = 8.58. The treatments that demonstrated stimulatory effects on
the growth of Lactobacillus acidophilus NCFM include run 12 (25g of green banana peel powder,
30g of carrot bagasse powder and 25g of orange peel powder), run1 (25g of green banana peel
powder, 40g of carrot bagasse powder and 25g of orange peel powder), run 17 (25g of green banana
peel powder, 35g of carrot bagasse powder and 20g of orange peel powder) and run15(20g of green
banana peel powder, 35g of carrot bagasse powder and 25g of orange peel powder) had a good
prebiotic response on Lactobacillus acidophilus NCFM. Run 10 had a high crude fibre content (1.91
percent), making it a stronger prebiotic option. Using Lactobacillus fermentum, Sreenivas and Lele
(2013) investigated the prebiotic activity of gourd family vegetable fibres. They discovered that
vegetables from the gourd family were high in prebiotics. The growth of probiotics was boosted by
composite powder in this study.Petkova and Denev (2013) reported that fruits and tubers had high
content of both prebiotic fructo-oligosaccharrides and inulin.

62
Table 4.6: Log colony forming unit of probiotic bacteria in composite powder

Log colony forming unit per ml. (log10 CFU/Ml) after 48 hours

Dilution factors for Bifidobacterium lactis Bi-07 and was Lactobacillus acidophilus NCFM 104 and
105 and 105 and 106 respectively.

Sample Bifidobacterium lactis Bi-07 Lactobacillus acidophilus NCFM

s
1 7.12 8.07
2 7.09 7.95
3 6.05 8.03
4 6.03 8.01
5 6.15 7.10
6 6.12 6.99
7 7.08 7.92
8 6.02 6.98
9 6.97 7.88
10 7.14 8.12
11 6.13 7.10

63
 12 7.10 8.06
13 6.04 8.00
14 6.16 7.18
15 6.05 8.02
16 6.04 8.01
17 6.05 8.03
18 7.06 7.90
4.7Sensory evaluation
The aroma, color, flavor, taste, and overall acceptability of the eighteen prepared soup formulations,
as well as the control sample (vegetable soup), were assessed (table 4.7). The control sample
received the highest grade for taste (8.6±0.51), followed by Run 2, 3, 9, 10, and 18 (5.6±0.84). Runs
5, 7, and 10 had the lowest taste score of (3.20.84) and were discovered to be dark in hue. This could
be owing to the addition of more banana peel to the soup mixture. Flavor (8.8±0.42) had the highest
score in runs 1, 2, 10, and 12, followed by control (8.1±0.56). Runs 5, 6, and 8 had lower scores.
This could be due to the decreased proportion of carrot bagasse powder. The overall acceptability
scores (8.4±0.69) were highest for control soup A0 and on this basis it was optimized.
Table 4.7 shows the mean hedonic scores achieved for composite powder soup preparation. Table
4.8 shows the results of the regression analyses for composite powder made with blends of green
banana peel powder, carrot bagasse powder, and orange peel powder, including coefficient estimates,
adjusted regression coefficients (R2 adj), and model significance and lack of fit for all sensory
attributes evaluated. The quadratic model proved effective in predicting the scent of the prepared
composite powder soup. Minitab version 18.1 software was used to create the regression model: (A
represents green banana peel powder, B represents carrot bagasse powder, and C represents orange
peel powder.)
Y= 0.1125A - 0.1000B - 0.2875C - 0.4750AB - 0.5000AC + 0.0750BC + 1.15A 2 - 0.1250B2 -
0.4000C2
The model could explain the observed variations and it did not present a significant lack of fit (p >
0.05). The regression model obtained for the Color of the composite powder soup was:
Y = 0.0875A + 0.3875B + 0.1750C + 0.9000AB + 0.2250AC - 0.1250BC + 0.6893A 2 - 0.2107B2 -
0.2357C2
The linear terms had a substantial impact on the color of the soup. According to the calculation, the
carrot bagasse powder produced the greatest improvement in Color sensory scores. Then came
orange peel powder and green banana peel powder, in that order. Even though the R 2 adj was less
than 60%, the projected Color model could be generated reliably based on the probability of F and

64
lack of fit values (Yusnita et al., 2007). The following is the equation derived from the data for the
flavor of the soups:
Y = 0.2000A + 0.4000B + 0.1500C + 0.6250AB + 0.2750AC - 0.0250BC + 0.6375A 2 - 0.2125B2 -
0.2625C2
The quadratic model for flavor data was significant (p< 0.05) in the multiple regression analysis and
could explain all variance in the hedonic results. The lack of fit test revealed that the model error and
replicate error were both minor, confirming the model's fitness for prediction. The taste scores were
statistically different, and it was discovered that adding green banana peel powder to the mix resulted
in lower hedonic scores (Table 3). The difference between the vegetable soup and the control was
not statistically significant (p< 0.05). The quadratic model was significant (p <0.05) in the design
expert application on taste scores, and there was no lack of fit. As a result, the variation was caused
by factors that were not considered in the model. The model obtained for the taste of the soups was:
Y = -0.3875A - 0.5000B + 0.1625C + 0.4500AB + 0.3250AC - 0.6000BC + 0.9232A 2 + 1.2000B2
- 0.1268C2
The regression model obtained for the overall acceptability of the soups was:
Y = -0.0250A + 0.0375B - 0.0375C + 0.3250AB - 0.0250AC + 0.1500BC + 0.8857A 2 + 0.0607B2
- 0.2393C2
The quadratic model was found to be non-significant in predicting the soups' general acceptability (p
> 0.05) in statistical analysis. The model was able to explain a large portion of the observed
fluctuations and showed no major lack of fit.

Table 4.7: The Mean ± Standard Deviation Value for Sensory Evaluation.

Run Aroma Color Flavor Taste Overall

acceptability
A0 8.0±0.81 7.2±1.07 8.1±0.56 8.6±0.51 8.4±0.69
1 8.8±1.03 7.3±0.82 8.8±0.42 4.4±0.84 7.8±0.78
2 6.8±1.03 7.3±0.82 8.8±0.42 5.6±0.84 7.4±0.69
3 6.1±0.87 5.9±0.56 7.3±0.48 5.6±0.84 6.5±0.52
4 6.0±1.05 5.9±0.56 7.3±0.48 4.5±0.84 6.5±0.52
5 5.2±1.13 4.7±1.15 6.5±1.08 3.2±0.84 5.7±0.94
6 5.3±0.67 4.8±1.03 6.5±0.70 4.5±0.91 5.7±0.82
7 5.2±0.78 5.3±1.05 6.7±1.05 3.2 ±0.82 5.7±0.94
8 5.4±0.96 4.8±0.78 6.5±0.70 4.3±0.91 6.2±0.78

65
 9 5.1±0.73 5.2±0.91 6.6±0.96 5.6±0.84 5.9±0.99
10 6.8±1.03 7.3±0.82 8.8±0.42 3.2±0.84 7.5±0.52
11 7.7±1.05 4.8±0.78 6.7±0.82 5.6±0.91 7.0±0.66
12 5.5±1.17 7.3±0.82 8.8±0.42 4.4±0.84 7.2±0.78
13 6.1±0.87 5.9±0.56 7.3±0.48 4.4±0.84 6.8±0.42
14 8.8±1.03 6.3±1.05 7.7±0.48 4.4±0.84 7.6±0.51
15 6.1±0.87 5.8±0.63 7.3±0.48 4.5±0.84 6.9±0.56
16 6.1±0.87 5.8±0.63 7.3±0.48 4.5±0.84 6.8±0.63
17 6.1±0.87 5.8±0.63 7.2±0.63 4.5±0.84 6.8±0.63
18 7.7±1.05 6.1±0.87 7.7±0.48 5.6±0.84 7.3±0.67

Table 4.8: Lack of fit, adj R2 and model (prob>F) of sensory evaluation
Variables Aroma Color Flavor Taste Overall
acceptability
A 0.1125 0.0875 0.2000 -0.3875 -0.0250
B -0.1000 0.3875 0.4000 -0.5000 0.0375
C -0.2875 0.1750 0.1500 0.1625 -0.0375
AB -0.4750 0.9000 0.6250 0.4500 0.3250
AC -0.5000 0.2250 0.2750 0.3250 -0.0250
BC 0.0750 -0.1250 -0.0250 -0.6000 0.1500
A2 1.15 0.6893 0.6375 0.9232 0.8857
B2 -0.1250 -0.2107 -0.2125 1.2000 0.0607
C2 -0.4000 -0.2357 -0.2625 -0.1268 -0.2393
Model 0.0425 0.0025 0.0059 0.0392 0.0920
(prob>F
Adj R2 -0.3046 -0.0563 -0.0851 0.3374 -0.1906
Lack of Fit 0.3349 0.0060 0.8643 0.0606 0.0521

4.8 Optimization
Numerical optimization suggested that soup made with 20.002g of green banana peel powder,
30.000g of carrot bagasse powder, and 15.168g of orange peel powder achieved the best solution
for this combination of variables after simultaneously maximizing, minimizing, and targetin
sensory quality similar to that of the control. The choice of root and fruit blends as the optimum
solution backs up an FAO report from 1990, which indicated that the fiber of roots and fruits (such
as oranges) is supplemented by that of other roots and fruits since they have a high crude fiber
content. The closeness of most observed and anticipated values showed that the matching models
were suitable. Even though a significant difference existed between the observed and predicted
values for taste, the observed value was still very acceptable.

66
 6) CONCLUSION AND RECOMMENDATION
6.1) Conclusion
Based on the findings of this study, the proximate composition for moisture, protein and crude fat
was high in Run 1 with 25g of green banana peel powder, 40g of carrot bagasse powder and 25g of
orange peel powder while it was low in Run 9 with 20g of green banana powder, 30g of carrot
bagasse and 20g of orange peel for moisture and Run 6 with 25g of green banana peel powder, 30g
of carrot bagasse and 15g of orange peel powder for protein and crude fat. The ash and crude fiber
was high in Run 10 with 30g of green banana peel powder, 35g of carrot bagasse powder and 25g of
orange peel powder. Generally the proximate analysis reveals that the composite powder had better
value than other study reported with single raw materials as per result and discussion. The results of

67
other Engineering properties was high for swelling capacity, bulk density, water holding capacity
and oil holding capacity in Run 5, 1, 18 and 7 respectively. The extracted inulin content from
composite powder of green banana peel powder, carrot bagasse powder and orange peel powder was
high in Run 10. Overall the distribution of inulin in formulated composite powder is nearly better as
compared with single inulin powder. As revealed from prebiotic properties of composite powder Run
1 had high resistance to acidic digestion while Run 6 had less resistant. Run 1 had high hydrogen per
oxide scavenging and antioxidant capacity of 91.4% and 81.4% respectively. The probiotic

propagation in Run 10 was 7.14 and 8.12 log CFU/ml which was high for both Bifidobacterium
lactis Bi-07 and Lactobacillus acidophilus NCFM respectively. Composite powder has low sensory
rating with respect to taste, and color. The sensory ratings of taste and color were negatively
influenced by sun dried green banana peel. The quadratic model for flavor data was significant (p<
0.05) in the multiple regression analysis and could explain all variance in the hedonic results. The
quadratic model was found to be non-significant in predicting the soups' general acceptability (p >
0.05) in statistical analysis. The model was able to explain a large portion of the observed
fluctuations and showed no major lack of fit.

6.2) Recommendation
Recommendations for future research based on the findings of the development and evaluation of the
green banana peel, carrot bagasse, and orange peel composite powder as prebiotic food ingredient of
the study include: Include the value analysis studies for developed green banana peel powder, carrot
bagasse powder and orange peel powder, developing a product with value added ingredients, which
may give some organoleptic changes, formulation and evaluation of products based on HACCP
procedures, determining the shelf life period using food analysis and various types of packaging
techniques, developing the marketing plan for new product in to food service system.

68
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Appendix 1: Laboratory Experiment figure Samples.

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Figure 1.1: Preparing composite powder and individual powder from raw material.

81
Figure 1.2: Determining technological properties of composite powder

Figure 1.3: Slicing the raw materials peel to produce powder.

82
Figure 1.4: Probiotic bacteria growth experiment.

83
Figure 1.5: Extraction of inulin from composite powder experiment

84
Figure 1.6: The purified and directly extracted inulin powder.

85
Figure 1.7: Antioxidant capacity determining of composite powder experiment.

86
Figure 1.8: Proximate analysis experiment.

87
Figure 1.9: Acidic digestion of composite powder

88
Figure 1.10: Sensory evaluation of composite powder soup

89
90
91
Figure 1.11: The scores of sensory evaluation of composite powder soup.

Appendix 2 ANOVA Tables for Sensory Evaluation

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Appendix 2.1: ANOVA Table for Aroma

Source Sum of Df Mean F-value P-value

squares square
Model 9.01 9 1.00 0.5330 0.0425 Significant

A-green banana 0.1013 1 0.1013 0.0539 0.0375

peel powder
B-Carrot bagasse 0.0800 1 0.0800 0.0426 0.5675
powder
C-orange peel 0.6613 1 0.6613 0.3522 0.5056
powder
AB 0.9025 1 0.9025 0.4807 0.0479

AC 1.00 1 1.00 0.5326 0.4841

BC 0.0225 1 0.0225 0.0120 0.0295

A2 5.92 1 5.92 3.16 0.0021

B2 0.0700 1 0.0700 0.0373 0.0052

C2 0.7168 1 0.7168 0.3818 0.5520

Residual 16.90 9 1.88

Lack of Fit 6.92 3 2.31 1.39 0.3349 Non-

significant
Pure Error 9.98 6 1.66

Cor Total 25.90 18

Appendix 2.2: ANOVA Table for Color

Source Sum of Df Mean F-value P-value

squares square
Model 7.35 9 0.8167 0.8934 0.0025 Significant

A-green banana 0.0613 1 0.0613 0.0670 0.0072

peel powder
B-Carrot bagasse 1.20 1 1.20 1.31 0.0055
powder
C-orange peel 0.2450 1 0.2450 0.2680 0.3056
powder
AB 3.24 1 3.24 3.54 0.0279

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AC 0.2025 1 0.2025 0.2215 0.4841

BC 0.0625 1 0.0625 0.0684 0.0495

A2 2.13 1 2.13 2.33 0.4521

B2 0.1989 1 0.1989 0.2176 0.0702

C2 0.2489 1 0.2489 0.2723 0.5520

Residual 8.23 9 0.9141

Lack of Fit 1.29 3 0.4308 0.3728 0.0060 Significant

Pure Error 6.93 6 1.16

Cor Total 15.58 18

Appendix 2.3: ANOVA Table for Flavor

Source Sum of Df Mean F-value P-value

squares square
Model 5.75 9 0.6385 0.8431 0.0059 Significant

A-green banana 0.3200 1 0.3200 0.4226 0.0032

peel powder
B-Carrot bagasse 1.28 1 1.28 1.69 0.0073
powder
C-orange peel 0.1800 1 0.1800 0.2377 0.4036
powder
AB 1.56 1 1.56 2.06 0.0400

AC 0.3025 1 0.3025 0.3995 0.5821

BC 0.0025 1 0.0025 0.0033 0.0411

A2 1.82 1 1.82 2.40 0.0854

B2 0.2023 1 0.2023 0.2672 0.0278

C2 0.3087 1 0.3087 0.4077 0.7430

Residual 6.82 9 0.7572

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Lack of Fit 0.7350 3 0.2450 0.2418 0.8643 Non-
Significant
Pure Error 6.08 6 1.01

Cor Total 12.56 18

Appendix 2.4: ANOVA Table for Taste

Source Sum of Df Mean F-value P-value

squares square
Model 17.81 9 1.98 2.02 0.0392 Significant

A-green banana 1.20 1 1.20 1.22 0.0425

peel powder
B-Carrot bagasse 2.00 1 2.00 2.04 0.0673
powder
C-orange peel 0.2113 1 0.2113 0.2154 0.5030
powder
AB 0.8100 1 0.8100 0.8259 0.0294

AC 0.4225 1 0.4225 0.4308 0.0485

BC 1.44 1 1.44 1.47 0.6102

A2 3.82 1 3.82 3.89 0.0501

B2 6.43 1 6.43 6.56 0.0820

C2 0.0720 1 0.0720 0.0734 0.0490

Residual 8.83 9 0.9808

Lack of Fit 6.03 3 2.01 4.32 0.0606 Non-

Significant
Pure Error 2.79 6 0.4657

Cor Total 26.64 18

95
Appendix 2.5: ANOVA Table for Overall Acceptability
Source Sum of Df Mean F-value P-value
squares square
Model 4.24 9 0.4711 0.6798 0.0920 Non-
Significant
A-green banana 0.0050 1 0.0050 0.0072 0.0307
peel powder
B-Carrot bagasse 0.0112 1 0.0112 0.0162 0.0483
powder
C-orange peel 0.0112 1 0.0112 0.0162 0.0502
powder
AB 0.4225 1 0.4225 0.6097 0.0940

AC 0.0025 1 0.0025 0.0036 0.0209

BC 0.0900 1 3.510.0900 0.1299 0.0612

A2 3.51 1 3.51 5.07 0.0429

B2 0.0165 1 0.0165 0.0238 0.0753

C2 0.2565 1 0.2565 0.03702 0.0390

Residual 6.24 9 0.6930

Lack of Fit 1.54 3 0.5142 0.6572 0.0521 Non-

Significant
Pure Error 4.69 6 0.7824

Cor Total 10.48 18

96