2.

Review of Literature

2.1 Originality and Distribution of Black mulberry

Black mulberry in family Moraceae, covers a vast range of Asia, Europe, North America, South
America and Africa continent and is widely cultivated in the Mediterranean and Middle Eastern
countries which are located at the junction of the Middle East and Mediterranean region, among
which Turkey has important black mulberry populations and the cultivation of these berries has
been appreciated for more than 400 years ago (Huang et al., 2013).

However, in Pakistan, black mulberry is suitably cultivated in colder regions such as Chitral,
Quetta, Azad Jammu and Kashmir, Miran Shah and in warmer ranges like Hyderabad Division,
covering an area of 459 hectares with total fruit production of 2100 tonnes annually. Moreover,
in Punjab the area covered by black mulberry is 208 hectares with fruit yield of 987 tonnes and
in Sindh, the cultivating area is 48 hectares with fruit production of 112 tonnes and in Khyber
Pakhtunkhwa, the covered area of land is 203 hectares with fruit production of 1001 tonnes.
These berries come into flower from April to May, and fruit ripens from July to September
(Aiman et al., 2018).

2.2 Fruit description

This dark coloured edible fruit is fully loaded with juice content and has a sweet taste with some
sourness that is more prominent feature in the less mature fruits and it is a multifeatured plant
and has greater economic value other than sericulture due to its many distinctive traits
(Skrovankova et al., 2015).

The ethnobotanical manipulation of black mulberry summarizes the utilization of fully ripened
fruits which are tremendously admired for their appetizing flavour and are consumed either as
fresh or after being processed (Krishna et al., 2013).

In the recent years, these kinds of berry fruits have achieved immense position in nutrition and
human health as they are remarkable source of principal phytonutrients specially antioxidants
and their effectiveness are directly dependent on the plant genotypes (Koyuncu et al., 2014).
In European countries, many research reports have inferred the chief presence of bioactive
components which has earned it ‘superfood’ status and it is predominantly grown for its fruits
(Natic et al., 2015).

Phytochemical composition

Plants are the ultimate source of many vital phytonutrients which have been considered essential
for the wellbeing of human beings. Food processing and health are interlinked factors that are
influenced by human dietary routine. All types of these berries consist of small edible fleshy
fruits that are regularly consumed all over the world not only in fresh and frozen forms but also
as processed products (Nile et al., 2014).

Black mulberry is an excellent source of many antioxidants such as resveratrol, zeaxanthin,

lutein and alpha and beta carotene that suggests the presence of functional constituents that are
advantageous for human health (Karlidag et al., 2012).

The black mulberry bioactivities such as antioxidant content, hypolipidemic effect and
macrophage activating effect are linked to the presence of phenolic substances with special
reference to bioactive compounds mainly due to its promising antioxidant status (Sofia et al.,
2014).

Nutritional composition

Fresh black mulberries are rich source of amino acids, vitamins and minerals such as zinc,
manganese, iron, potassium and calcium that play essential role in the human body. Potassium is
an important component of cell and body fluids that helps in controlling heart rate and blood
pressure. Manganese is used by the body as a co-factor for the antioxidant enzyme, superoxide
dismutase. High iron content, which is a rare feature among berries, contain s fruit and Iron,
being a component of hemoglobin inside the red blood cells, determines the oxygen-carrying
capacity of the blood. Calcium

In addition, black mulberries are also rich in pectin and fibrin and ascorbic acid content
are rich in B-complex group of vitamins and vitamin K. Contain very good amounts of vitamin
B-6, niacin, riboflavin and folic acid. These vitamins are function as co-factors and help body in
the metabolism of carbohydrates, protein, and fats.

Medicinal properties

Over the last years, berry fruits have moved under the spotlight of nutritional research since an
overwhelming body of research has highlighted their ability to impact human health and disease
prevention. The fruits of black mulberry are famous for their nutritional qualities, flavour and
especially medicinal properties (Bosnjakovic et al., 2012).

The juice from roots of black mulberry has the ability to reduce blood sugar in diabetic patients
and also has some effect on the pancreas and glycogenesis. Similarly, root bark contains calcium
malate, tannins, phytobaphenes, sugar, phytosterol, fatty acids and phosphoric acid and in other
studies links are also being made between high levels of antioxidants in the diet and protection
from some of the most distressing diseases of this present age –chronic diseases like
Alzheimer’s, many forms of cancer and Parkinson’s disease (Lee et al., 2013).

Highest amount of anthocyanins protects our body from the harmful oxidation of free radicals
specifically, mulberry contains cyaniding 3-glucoside, which epidemiological studies confirm
lowers the risk of many degenerative diseases such as chronic arthritis and atherosclerosis.
Cyaniding 3-glucoside protects the body against cardiovascular disease and diabetes and black
mulberry along with improving blood circulation can help people who suffer from heart
palpitations (Salcedo et al., 2015).

In traditional folk medicines mulberries are also used for efficiently treatment of fever, anemia,
sore thro and hypertension and consuming black mulberries during the hot season benefits in
blood disorders and has a cooling effect on the body. It enriches the blood in the process while
soothing and calming the nerves. It also helps in promoting the metabolism of alcohol. Eating
even a small bowl of black mulberries before sleeping can cure sleeping disorders. It can also
help someone suffering from anaemia and can aid suffering from vertigo. A few pieces of black
mulberry fruit can relieve constipation, lower blood pressure and improve the digestive system
(Kostic et al., 2013).
According to traditional Chinese medicine, black mulberry can protect from kidney and liver
damage, improve eyesight, strength the joints and have radio protective and anti-aging effects.
(Wattanathorn et al., 2012).

In Korea, black mulberry is used as a medicine because of anti-inflammatory, antibiotic,

antioxidant, and hepatoprotective properties and high level of antioxidants in black mulberries is
an excellent weapon to fight against infection like influenza virus (Kamiloglu et al., 2013).

Uses in food industry

Today many industries are investigating, generating and applying natural bioactive compounds
for the preparation of dietary supplements, nutraceutical, functional food ingredients or
cosmeceuticals and within this trend food technologists and food scientists are making efforts to
explore different parameters on the content of bioactive compounds and on the antioxidant
activity and have developed food products and food additives using natural bioactive compounds
(Arfan et al., 2012).

Fruit juice

Black mulberry fruit juice has been commercially produced as a health beverage, and it has
become very popular in China, Japan and Korea and is extremely beneficial for post-operative
patients. It accelerates healing, improves blood circulation, decrease swelling, aid recovery after
child birth and prevents the onset of infection. It can balance internal secretion, enhance the
immunity and promotes body fluid production. It also helps people who suffer from dehydration
(Krishna et al., 2015).

Fruit tea

It is natural, pleasantly savour all year-round, hot or cold. It does not contain caffeine and
therefore can be safely drunk at any time of the day. In Chinese markets, black mulberry is often
provided Jam is one of the most popular products made from in the form of a paste called
“Sangshengao”-mixed in hot water to make tea (Wang et al., 2013).

In Iran, dried black mulberries are used as a sweetener in black tea and after a sip of tea, dried
black mulberries are eaten to sweeten the mouth (Gundogdu et al., 2018).

Fruit syrup
It is a kind of molasses obtained from mulberries. It is very beneficial to be consumed as food by
anaemic patients. It is good especially for stomach illness and ulcer. It is used to increase body
resistance against cold, for asthma and bronchitis patients and also a source of energy for sports
persons. It helps intelligence development and physical development of babies and children.
During gargling, it is efficient for mouth and throat illness. It is suggested for the diet of pregnant
and breast feeding women, tuberculosis patients and those in healing period (Sujathamma et al.,
2015).

Fruit wine

Over ripened and sour fruits have high source of vitamin-C which is commonly used for the
preparation of special wine and beer in most of the cold countries. A glass of mulberry wine a
day helps to get rid of impurities in the body which can help and make the body slim. It is
believed that small dose of the drink protects against stomach and heart diseases and very
popular as a ladies drink in Europe (Alakbarov et al., 2015).

Jam Jelly

& marmalade

Jam is one of the most popular products made from to evaluate anthocyanins and total phenolics
content of mulberries jam and its colour development during storage at different temperature and
light regimes, as well as the influence of cultivar on jam production. Hepsağ

On the basis of various quality parameters, jelly with 50 per cent juice and 45 per cent sugar was
could be stored safely for a period of six months under both the ambient and refrigerated storage
conditions and also in both packaging materials like PET and glass jars Thakur Black mulberry
fruit powder could be used as ingredient to the production of delightful, nutritious and delicious
jellies. Anthocyanin-rich jelly with 13% mulberry fruit powder was found to be accepted by the
consumer. Moreover, the developed jelly was contained higher total phenolic contents,
antioxidant activities and total anthocyanins than the control formula. This development only
improved the nutrients of the product but it helped to increase the values of the ingredient and
the product (Tongmai et al., 2019).

Frozen desserts
Fruit powder

Raw black mulberry fruit powder is a true nutritional treasure, being used in both alternative and
traditional medicines for years. It contains a vast amount of nutrients essential to one’s health
with an overwhelming amount of anti-inflammatory, antiseptic cleansing qualities and healing
properties to refresh and restore the body. It promotes healthy liver and kidney function, and
improves digestion and assimilation (Liang et al., 2012).

Food color

Fruits can be used as colouring and flavouring agent as there is increasing demand for natural
food colorants, black mulberry fruit extract being water-soluble and easily extractable, acts as a
good colouring and flavouring agent due to abundant presence of anthocyanins which promise
greater antioxidant activity (Shivkumar et al., 2015).

Functional food products

Black mulberry is a highly nutritious fruit and can be efficiently used to propose novel and
functional food products. Such food products are improved descriptor of conservative foods
which assure different characters. Many functional food products have been designed by using
black mulberry as their constituent part such as syrups, tea, juices, jams & marmalades, frozen
desserts, ice-cream, pickle, paste, sauces, natural dyes and dried fruits (Krishna et al., 2015).

Sauces

Therefore, designer food product like sauce with a perfect combination of high acidity and low
moisture content was developed in the food industry and this processed food item has become
more prevalent among consumers due to its several features such as enhanced taste, appearance
and feasibility (Perera et al., 2017).

Several types of sauces are commonly a part of Asian culinary, carrying greater appeal to
different dishes and various kinds of fruits and vegetables along with added ingredients are used
in sauce making. Sauces are generally thinner and contain more total solids (minimum 30%) than
Ketchups (minimum 28%). These are typically packed in feasible packaging materials and can
be termed as convenience food products. The other advantage of sauces is their ability to
improve the taste of food. For example, hot sauces add piquant taste to different dishes, meats,
vegetables; sweet sauces grant attractiveness to pancakes, rice, desserts and ice-creams. Sweet
andsoursaucesfindapplicationinmeatdishes .Sauces are of two kinds: 1) Thin Sauces of low
viscosity consisting mainly of vinegar extract of flavoring materials like herbs and spices, and 2)
Thick sauces that are highly viscous. Sauces are prepared from more or less the same ingredients
and in the same manner as chutney, except that the fruit or vegetable pulp or juice used is sieved
after cooking to give a smooth consistency to the final product. However, cooking takes longer
because fine pulp or juice is used. Some sauces develop a characteristics flavour and aroma on
storing in wooden barrels. High quality sauces are prepared by maceration of spices, herbs, fruits
and vegetables in cold vinegar or by boiling them in vinegar. A fruit sauce should be cooked to
such a consistency that it can be freely poured without the fresh tissues separating out in the
bottle. The color of the sauce should be bright. Sauces usually thicken slightly on cooling. By
using a funnel hot sauce is filled in bottles leaving a 2cm. head space at the top and the bottle are
sealed or corked at once. The necks of the bottles, when cold, are dipped in paraffin wax for
airtight sealing. It is advisable to pasteurize sauces after bottling since there is always a danger of
fermentation, especially in tomato and mushroom based Sauces. Other sauces are more acidic
and less likely to ferment but should be pasteurize all the same. For this the bottles are kept in
boiling water for about in 30 min Thus, the use of sauces in our daily life is increasing
continuously due to their nutritional prominence and storage stability (Gundogdu et al., 2018).

In this view, Kembayau fruit sauce was developed for determining compositional content and
physicochemical parameters (Rahman et al., 2015) with the estimated ranges of 60.43±0.40 %
moisture content, 0.50/ ±/ 0.10 % crude ash, 0.75 ± 0.03 % crude fat, 1.40 ± 0.07 % crude
protein, 0.92 ± 0.02 % crude fiber and of 35.50 ± 0.04 % carbohydrate.

In comparison, moisture content for mango sauce was 67.89±0.40% (Akhtar et al., 2009) and
moisture content of tomato sauce was 66.65±0.14 % (Sharoba et al., 2005) and 0.50±0.10 %
crude ash with an increased value was obtained, as compared to mango sauce having 0.38±0.01
% crude ash and low value of 2.94±0.05 % crude ash as compared to tomato sauce.

Kembayau sauce contained 0.75 ± 0.03 % crude fat, at higher value compared to tomato sauce
0.62±0.01 % and low amount /compared to mango sauce 0.89±0.02 %. Kembayau fruit sauce
contained 1.40 ± 0.0/7 /% of crude protein which is comparable with mango sauce and tomato
sauce with crude protein at 1.28±0.02 % and 1.04±0.03 % respectively.
During this investigation, it was reported that the total soluble solids of Kembayau fruit sauce
were increased for 1°Brix in comparison with total soluble solids for apple sauce which was
around 20.33±0.58 % (Katerson et al., 2002) and the total soluble solids for the tomato sauce
were about 33.35±0.14 % (Sharoba et al., 2005).

The pH value for Kembayau fruit sauce in the study was decreased about 0.05 % within eight
weeks of storage (Bajwa et al., 2008). According to previous reports, the set range of pH for
sauce is 2.3 to 4.0. Therefore, the Kembayau fruit sauce in study met the pH standard for sauce.
In comparison, the pH value for the apple sauce is 3.14±0.03 (Katerson et al., 2002) and the pH
value for tomato sauce is 3.48±0.02 % (Sharoba et al., 2005) and the titratable acidity value of
the Kembayau sauce is inverse to the pH value and this value 1.13±0.03 % is higher than tomato
sauce having 0.96 % titratable acidity value (Avinash et al., 2014).

The viscosity of the Kembayau sauce in the study was 24.30±0.09 % and the change in colour of
Kembayau sauce was in the range of 5.91±0.5 % during storage (Rahman et al., 2015) and the
best formulation of Kembayau sauce was studied for the varied sensory attributes also.

The nutritional prospective and great admiration of black mulberry aroused the investigations on
its composition involving its antioxidant capacity to discover novel sources of bioactive
compounds with improved metabolic activities.

Keeping in view all these considerations, the present exploration is intended to formulate black
mulberry sauce using black mulberry as a basic ingredient with enriched antioxidants. The final
product will be subjected to study various factors including compositional assay,
physicochemical aspects and sensory characters.

2.1 Compositional analysis

The study was held to assess quality characteristics of black mulberry sauce. The fresh black
mulberries were used for making sauce. The bioactive compound enriched black mulberries were
integrated in sauce formulation in variable ratios 100:0, 85:15 and 70:30.The product was
examined to find m/o/isture co/ntent, ash content, crude protein, crude fat and carbohydrate (Kim
et al., 2016).The moisture content, ash content, crude protein, crude fat and carbohydrate was %.
The average moisture content, ash content, crude protein, crude fat and carbohydrate were found
to be 80.14±0.05 g.100g-1 (fresh weight) and others as g.100g⁻ 1(dry weight) 3.92±0.01,
10.34±0.04, 1.93±0.06 and 75.58±0.54 respectively as per the standard methods adopted by
Sadia et al. (2014). The calorific value was estimated as 361.1±0.98 Kcal.100g-1.

The compositional analysis of fresh black mulberry including moisture content, ash content,
crude protein, crude fat and carbohydrate was found to be 81.6 % ,0.24 %,10.29 %,5.75% and 6-
9% respectively as per the protocol used by Koyuncu et al .(2014).

The moisture contents in mulberry fruits were in the range of 85.9 ± 0.4 to 80.6 ± 0.6 %. The
results for Bulgarian species were near to the reported data for the moisture contents in Morus
species (Pakistan12 and Macedonian27 origin) 78 - 82 % and higher from these from Turkish
origin18- 71.5% to 74.6%. The moisture content in leaves were 68 - 74 %. The lowest values for
leaves and fruits were obtained for M. rubra – 68 % and 81 %, respectively.

The plant samples were assessed for moisture, ash, crude fat, crude protein and fiber on a dry
weight basis, according to AOAC (1990). The moisture was measured in a drying oven at 105°C
until constant weight. Analysis of crude fat was done in a Soxtec system HT (Tecator) using
petroleum ether (bp. 40-60°C) Determination of crude protein (% N x 6.25) was accomplished
by employing micro-Kjeldahl method. Crude fiber was estimated by treating the samples with
acid and alkali using Fibertec system (Tecator) and ash contents measured by heating the
samples at 550°C. Carbohydra//tes (%) c/ontents were estimated by using a difference method,
by deducting the sum of the percent of moisture, fat, protein, ash and fiber from 100. The
calorific value was measured by multiplying the values of carbohydrate, lipid, and protein with
the factors 4, 9 and 4, respectively. Then the products were summed and expressed in kcal per
100 g. The assessment of ascorbic acid was carried out with 2,6-dichlorophenol-indophenol
using titration method. The proximate compositions of black mulberry fruits collected from
various geographic locations are given in table 1. The moisture content of the fruits was in the
range of 8.9 to 12.3%. The highest moisture value was recorded for the sample collected from
Swat and lowest for the sample acquired from Swabi. The ash contents were found different at
the significance level of p<0.05 for all the tested fruits. The ash values ranged between 7.0
(sample from Swat) to 9.8% (sample from Peshawar). The an//alysis of t/he crude fats, indicated
the concentrations between 2.7 to 4.7%. The sample acquired from Swabi showed the maximum
value. In case of protein contents, the samples collected from D. I. Khan showed the highest
value (13.1%), followed by that of the samples collected from Peshawar (11.1%), Swat (9.4%),
Karak (9.3%) and Swabi (8.6%). The fibre contents of the fruits varied significantly (p<0.05)
with respect to collection points. The fruits collected from Swabi showed highest fibre content
(14.4%). The carbohydrate contents depicted significant variations in their concentrations
(p<0.05) with reference to sampling sites. Highest value was observed for the sample collected
from Swat. The calorific values, calculated on a dry weight basis, ranged between 278.8 to 297.1
kcal/100g. However, these values were found statistically (p>0.05) the same for all the samples.

Total phenolic content

Total phenolic content of resultant sauce was found to be Sofia et al. (2014) conducted a study to
estimate the total phenolic content of black-coloured mulberry stored in polythene bags in frozen
state and the estimated value was found to be 1.02 mM GAE/100 g fw. The phenolic content of
the Morus nigra fruits ranged between 558.0 to 1090.7mg of gallic acid equivalent (GAE) per
100g. Earlier, Khalid et al. (2011) reported 2050 µg/g phenolic contents in the fresh juice of
black mulberry, whereas a study conducted by Özgen et al. (2009) showed 2737 µg/g phenolics
in Morus nigra fruits. Mahmood et al. (2012) reported 575, 1722 and 2287 mg/100 g phenolics in
unripen, semi ripened and fully ripened samples of Morus nigra, respectively. The highest
quantity of total phenols was found in the sample collected from D. I. Khan

Total phenol cont//ents of th/e extracts were determined by the modified Folin-Ciocalteu method
[12]. An aliquot of the extracts (1 ml) was mixed with 0.5 ml of Folin-Ciocalteu reagent and 2 ml
of sodium carbonate (20 %). Absorbance was measured after 10 min incubation at room
temperature at 760 nm. Total phenolic content was expressed as mg/100 g gallic acid equivalent
(GAE). The result of each one assay was obtained from three parallel determinations.

Dimitrova et al. (2015) also conducted a study to determine total phenolic content of fresh black
mulberry fruits and the calculated value ranges from 17.66 to 34.88.

Khattak et al. (2015) also studied the total phenolic content of fresh black mulberry fruits and the
calculated values ranged bet/ween 558.0 to 1090.7mg of gallic acid equivalent (GAE) per 100g.
Kostic et al. (20//19) also conducted a study to evaluate total phenolic content of fresh black
mulberry ranges for maceration and ultrasonic extractions from 60.04 to 150.13 mg and 69.11 to
142.18 GAE/100g fresh fruit, respectively.

Total phenolic content (TPC) was determined by the Folin– Ciocalteu′s reagent method adapted
to microscale (Mena, Martí, Saura, Valero, & García-Viguera, 2013). TPC was evaluated by
measuring the variation in absorbance at 765 nm after 1 h of reaction. Assays were measured by
using 96-well microplates (Nunc,Roskilde,Denmark) and Infinite® M200microplatereader
(Tecan,Grödig,Austria). Results were expressed as mg of gallic acid equivalents (GAE) per g of
dry material.

2.2 Total flavonoid content

Mahmood et al. (2012) reported 245, 706 and 1021mg/100g total flavonoid content in unripen,
semi ripened and fully ripened samples of black mulberry fruits taken from different localities of
Khyber Pakhtunkhwa, Pakistan, respectively.

Khattak et al. (2015) conducted a study to check total flavonoid content in black mulberry fruits
analysing, different samples and the highest content was recorded for sample collected from
Swat that ranged from 63.7 to 244.0 mg/100g. The samples obtained from Karak and Swabi with
135.0 and 147.7mg/100g flavonoids were found statistically equal. Stojanovic et al. (2016) also
conducted a study to estimate the total flavonoid content as catechin (mg CE/100g) using the
equation based on the calib/ration curve and the calculated value was ranged from 60.02 to 82.08
and from 53.13 t//o 108.16 mg CE/100g, respectively.

Flavonoids contents were assessed by employing the method of Aiyegoro and Okoh (2010). One
ml of the fruit extract was mixed with 0.2ml of 10% aluminum chloride, 0.2ml of 1M potassium
acetate, 3ml of methanol and 5.6ml of distilled water. The mixture was kept at room temperature
for half an hour. The absorbance of the mixture was noted at 420 nm. Quercetin was employed
as standard (0-1mg/ml). Concentration levels of flavonoids were calculated from the standard
curve and reported as mg/100g quercetin equivalents.

Flavonoid contents of the fruits of the samples varied from 63.7 to 244.0 mg/100g. Previously,
Mahmood et al. (2012) rep/orted 245, 706 and 1021mg/100g flavonoids in unripen, semi ripened
and fully ripened samples of Morus nigra, respectively. Yang et al. (2010) reported 0.39% total
flavonoids in M//orus alba fruit. Highest content was recorded for sample collected from Swat.
The samples obtained from Karak and Swabi with 135.0 and 147.7mg/100g flavonoids were
found statistically equal.

2.3 Total anthocyanins

The black mulberry sauce was subjected to estimate total anthocyanins as per the standard
protocol of Liang et al. (2015) and the estimated value was found to be

Kamiloglu et al. (2012) also conducted a study to estimate the total anthocyanin content in fresh
black mulberry fruits and estimated range was expressed as 1221.0±42.9 milligrams of cyanidin-
3-O-glucoside (C3G) equivalent per 100g of dw of sample.

Kostic et al. (2019) conducted a study to estimate total anthocyanin content of fresh black
mulberry using the process of maceration and the estimated range was from 52.03 to 137.06 for
maceration and from 64.08 to 137.06 mg Cy-3-O-glu/100 g for ultrasonic extraction of black
mulberry.

Know et al. (2019) conducted a study to estimate the total anthocyanins in black mulberry made
up of flavonols such as kaempferol and quercetin, and the estimated value was reported as
27mg/10g of fresh berries with cyanidin 3-O-glucoside being the major anthocyanin (66.3%),
followed by cyanidin 3-O-rutinoside (27.8%), pelargonidin 3-O-glucoside (4.4%), and
pelargonidin 3-O-rutinoside (1.5%).

The total monomeric antho/cyanin content was evaluated by a pH differential method (Giusti and
Wrolstad, 2001). Absorbance was recorded with a UV-visible spectrophotometer at 510 and
700nm at pH 1//.0 and 4.5 employing the following equation. A= (A510nm−A700nm) pH1.0-
(A510nm−A700nm) pH4.5

Data were calculated with the extinction coefficient for cyanidin-3-glucoside (ε=29,600) and the
results are described as mg cyanidin-3-glucoside per 100g dry weight.

The analysis of anthocyanins depicted significant variations with reference to the geographical
origins of the plant. The anthocyanins ranged from 67.0 to 346.3 mg of cyanidin 3-glucoside
equivalent per 100g on dry weight basis. Highest content were observed for sample picked from
Swat and lowest for sam/ple of Peshawar. Earlier research conducted by Ercisli et al. (2010)
showed that the black mulberry contained 719 µg/g of anthocyanin content on fresh mass basis.
While, a study carried o//ut on Turkish mulberry species (Özgen et al., 2009) indicated that black
mulberry had the highest amount of anthocyanins (571µg/g). The alkaloids ranged between
404.0 to 648.3 mg/100 g on dry weight basis. Previously, Imran et al. (2010) analysed the fruits
of four Morus species and found that Morus nigra contained 630mg/100 g of alkaloid contents.

2.4 Antioxidant activity

Mena et al. (2011) conducted a study to check antioxidant activity of fresh black mulberry using
ABTS assay and calculated mean values varied significantly from 3.84 to 20.73 mg trolox g dw
and from 3.62 to 12.91 mg trolox g dw using DPPH assay.

The free radical scavenging activity was determined using ABTS•+ and DPPH• assays according to Mena
et al. (2011).Assays were measured by using 96-well microplates (Nunc, Roskilde, Denmark) and
Infinite® M200 micro plate reader (Tecan,Grödig, Austria). Results were expressed as mg trolox
equivalents per g of dry material

Iqbal et al. (2010) conducted a study on black, white and red mulberries in Pakistan and
identified total antioxidant value using ABTS assay, as 4.4913.99 µmolTE g1 and they reported
that black mulberries contained more antioxidant capacity than white mulberries.

Similarly, according to Khalid et al. (2011) conducted a study on black mulberries using ABTS
assay and had total antioxidant value as 1420 µmolTE g1. Compared to our study, Gundogdu et
al. (2018) measured h/igher antioxidant value using ABTS assay in black mulberries as 25.55
µmolTE g-1.

The DPPH ra//dical-scavenging ability Each 70 % ethanol extract of mulberry leaves and fruits
(0.15 ml) was mixed with 2.85 ml freshly prepared 0.1mM solution of DPPH in methanol. The
sample was incubated for 15 min at 37 °C in darkness. The reduction of absorbance at 517 nm
was measured by spectrophotometer in comparison to the blank containing methanol and %
inhibition were calculated36. A standard curve was built with 6-hydroxy-
2,5,7,8tetramethylchroman- 2- carboxylic acid (Trolox) in concentration between 0.005 and 1.0
mM. The results were expressed in mM Trolox® equivalents (TE) per g fresh weight.

The scavenging activity of the fruit samples was determined using 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical (Kha/ttak, 2012). Two ml of DPPH radical in methanol solution (60µM) was
mixed with eighty µl of the sample and shaken appropriately. The solution was incubated in dark
at 37°C for an hour a///nd then its absorbance was measured at 517nm. The absorbance of
corresponding blank (control) was also noted. The results were expressed as EC50 values. EC50
value is defined as the extract concentration at which DPPH radicals were reduced by 50%and it
is estimated from the linear regression analysis.

The ABTS assay was performed as described by Re et al. (1999). The stock solutions included 7
mM ABTS solution and 140 mM potassium persulfate (PPS) solution, and the two stock
solutions were mixed in equal quantities for 12 h at room temperature in dark. The solution was
diluted by mixing 1 mL ABTS+ solution with 60mL of 80% ethanol to obtain an absorbance of
0.7 units at 734 nm using a spectrophotometer. The mulberry extracts (1 mL) were allowed to
react with 1 mL of the ABTS+ solution, and after 7 min the absorbance was taken at 734 nm.
The scavenging activity of ABTS radicals from the mulberry extracts was compared with that of
BHT. The percentage inhibition was calculated as follows:

ABTS radical-scavenging activity (%)={1−(A/B)}×100

where A is the absorbance of the sample treated with the extract, and B is the absorbance of the
untreated sample.

pH and Titeratable acidity

The total acidity was determined according to the AOAC method (Anon., 2003). The total
acidity present in fresh mul/berry juice averaged 1.60%, which is a little higher then has been
reported for red and white mulberries (Ercisli et al., 2010) and for black mulberry (Iqbal et al.,
2010).

For the estim///ation of titratable acidity and pH, the fruits were squeezed and filtered. Fifteen ml
of the juice was diluted with 35ml of distilled water in a 50ml flask. The solutions were stirred at
200 rpm on magnetic stirrer for half an hour at 25°C and then filtered. The pH values were
determined in triplicate using a Microcomputer pH Vision Datalogger (6091, JENCO electronic
Ltd., China) at room temperature. Three point calibrations were performed with pH 7.0, 4.0 and
2.0 buffers (Fischer Scientific). For the determination of titratable acidity, 5 ml of the solution
was taken in a 25ml flask and then2 to 3 drops of phenolphthalein (1%) were added.
Subsequently this was titrated with 0./1N sodium hydroxide.
The jelly s///howed a slight decrease in titratable acidity during storage (Fig. 2c) which was
comparatively higher under ambient conditions as compared to the refrigerated conditions due to
the faster rate of reactions as a result of high temperature in ambient conditions. The decrease in
titratable acidity might be due to co-polymerization of organic acids with sugars and amino
acids. Similar results were recorded by Singh et al. (1985) in guava jelly and Relekar et al.
(2011) in sapota jelly.

The pH values and titratable acidity of the fruit samples are presented in fig. 1. The pH values of
the selected samples were in the range of 3.4 to 4.9. The sample collected from D. I. Khan
showed the highest pH, followed by that of the Peshawar and Karak. The pH values (3.52-5.60)
reported earlier by Ercisli and Orhan (2007) are in good agreement with the present findings. The
titratable acidity of the tested samples ranged from 0.9 to 1.7%. Significant variations (p<0.05)
were noted for acidity values for all samples except for the fruit grown in Swat and Peshawar.
These results are in line with previous findings reported by Imran et al. (2010), which showed
titratable acidity ranging from 0.84 to 2.00% for the fruits of Morus species

The pH value for Kembayau fruit sauce in the study was decreased about 0.05 within eight
weeks of storage. The decrease value of pH is correlated with increasing acidity value that
commonly due to the present of lactic acid bacteria [24, 25]. Previous report, the range value of
pH for sauce is 2.3 to 4.0 [26], therefore the Kembayau fruit sauce in study were meet the pH
standard for sauce. In comparison, the pH value for their apple sauce is 3.14±0.03 , and the pH
value for tomato sauce is 3.48±0.02 [23]. /

Acidity val///ue of the Kembayau sauce is inverted proportionate with the pH value. Acidity
value increase is due to production of acid at low ion at low concentration during the storage.
Scientific report stated that, this acidity value is influenced by sodium benzoate that added into
product . The increasing of acidity value may be result from production of acid from
polysaccharide degradation and sugar oxidation, or through breakdown of pectin molecule in
sauce. In comparison, acidity value of apple sauce is 0.91±0.05 , and 1.54±0.03 for tomato sauce.

Total soluble solids

Total soluble solids were determined with a digital refractometer (Kyoto Electronics, Japan) by
using one drop of jui/ce in digital refractometer at 25°C and were expressed as a percentage of
fresh juice (volu///me basis). Total soluble solids in black mulberry juices averaged 19.40%; this
high content of soluble solids gave a thick appearance to black mulberry juice. Koyuncu et al.
(2004) reported 13.11 to 16.23% total soluble solids in black mulberry genotypes, and similarly,
Iqbal et al. (2010) reported 8.88% TSS in black mulberry fruits obtained from Khyber
Pakhtunkhwa area, Pakistan. The total soluble solids in mulberry grown in different agro-
ecological regions ranges from 15 to 31% (Lee et al., 2004; Ercisli et al., 2010; Iqbal et al.,
2010).

Vitamin C

Ascorbic acid content of jelly decreased significantly during storage; however, the decrease was
lower in refrigerated storage conditions than ambient conditions (Fig. 2e). Decrease in ascorbic
acid content might be due to its degradation into dehydro-ascorbic acid or furfural during
storage. Less decrease of ascorbic acid in refrigerated storage might be due to the slower rate of
its degradation in low temperature as compared to ambient storage conditions because of its
susceptibility to high temperature. The overall effect of storage conditions as well as packaging
materials on ascorbic acid content of jelly was found to be significant.

The assessment of ascorbic acid was carried out with 2,6-dichlorophenol-indophenol using
titration method. The data showed significant effect (p< 0.05) of collection points on vitamin C
levels. Maximum value was recorded for the sample collected from Swabi, while lowest for the
samples of Swat and Peshawar. A study performed by Iqbal et al. (2010) depicted 32mg/100 g
vitamin C content in fresh black mulberry fruit.

The pH value for K/embayau fruit sauce in the study was decreased about 0.05 within eight
weeks of storage///. The decrease value of pH is correlated with increasing acidity value that
commonly due to the present of lactic acid bacteria. Previous report, the range value of pH for
sauce is 2.3 to 4.0, therefore the Kembayau fruit sauce in study were meet the pH standard for
sauce. In comparison, the pH value for their apple sauce is 3.14±0.03 , and the pH value for
tomato sauce is 3.48±0.02. Acidity value of the Kembayau sauce is inverted proportionate with
the pH value. Acidity value increase is due to production of acid at low ion at low concentration
during the storage. Scientific report stated that, this acidity value is influenced by sodium
benzoate that added into product . The increasing of acidity value may be result from production
of acid from polysaccharide degradation and sugar oxidation, or through breakdown of pectin
molecule in sauce. In comparis/on, acidity value of apple sauce is 0.91±0.05, and 1.54±0.03 for
tomato sauce.

Viscosity///

The viscosity of the Kembayau sauce using viscometer in study was decreased in long period of
storage, but in small value from 0.03 - 0.12 centipoise. This is due to low usage of xanthan gum
in Kembayau sauce at 0.1%, even it suggested at 0.1 to 0.5%. Syneresis that occurs in sauce will
affect to the final viscosity of the sauce product [29]. The standard range of viscosity for the fruit
sauce is from 20 - 25 centipoise [26], therefore the Kembayau sauce in the study was meet the
viscosity requirement.

Color

colour was determined using Hunter Colorimeter. There is very minimal value of colour (L*, a*,
b*) deterioration during storage of Kembayau sauce. The L*=lightness represent scale from 0 to
100, whereby 0=darkness/black hue, and 100=lightness/white hue. Meanwhile, a* and b*
represent by negative and positive, whereby –a*=green hue; +a*=red hue; and –b*=blue hue, and
+b*=yellow hue. Colour deterioration of Kembayau sauce is due to natural pigment oxidation of
fruit puree, and it happened due to existence or present of oxygen [30]. There are two
possibilities of oxidation reaction happened in Kembayau fruit sauce: due to available oxygen (1)
during mixing of all raw materials, and (2) during storage. The normal accepted colour for the
sauce is in the range of light red to dark brown [32]. Therefore, Kembayau sauce in study can be
categorized as having light brown colour and most probably can be accepted by consumer.

Sensory response/

The best blended Sauce was selected by senso///ry evaluation which was conducted on 9 point
Hedonic Scale for different sensory attributes like appearance color, flavor and over all
acceptability by a panel of 20 semi- trained members having experience of sensory evaluation of
fruits and vegetable products. The Tomato and Bael fruit juice were blended in various ratios that
are 90:10, 80:20 and 70:30. The experimental Sauce prepared using Tomato and Bael fruit was
evaluated by sensory analysis. Mean scores obtained for the various proportions by sensory
analysis are given in Table 2:The sensory attributes „Colour and Appearance‟ of the Sauce made
from Tomato and Bael for 70:30 proportion i.e. for C treatmentnoted highest mean score 9.0
where as treatment „A‟ and „B‟ were not much acceptable by colour and appearance point of
view. The average scores forattribute „texture‟were noted to be maximum for treatment „C‟
followed by „B‟ and „A‟ treatments. Similarly mean score for another sensory attribute „Taste‟
noted highest again for treatment „C‟.For sample „A‟average score obtained for sensory
attribute „Taste‟was noted to be minimum and highest for sample „C‟ while for sensory attribute
„Flavor‟Minimum average scor/e observed to treatment „B‟ and highest for sample „C‟. The
sample „A‟ and „B‟ were almost unacceptabl///e because of decreasing concentrationof Bael
juice.Following remarks were obtained by the panelist for all three test samples: Sample
„C'obtained „Liked extreme/ely‟ remark, while Sample „A‟ received „Liked very much‟ and
„B‟got “Like Moderately” remarks respectively.Though Bael juiceisarichsource of nutrients but
it has not been accepted due to its poor taste and flavor. Blending tomato and Bael is good
options to improve the utilization of Bael. Similar kind of research was done by (Mishra and
Chopra, 2006) was prepared a mixfruit jam from Bael and mango in this final optimized jam had
45%mixed pulp ,70%T.S.S. and 0.5%acidity based on organoleptic characteristic[21]. Thus over
all acceptability of sample „A‟ and „B‟ was good but sample „C‟ received highest hedonic scale
score by all the panelist, therefore it wasselected for chemical analysis and large scale
production.
The consumer acceptability of the developed product was evaluated by a taste-testing panel
using 1-9 point Hedonic Rating Test (HRT). The panelist scores were analyzed with MSTAT-C
software.