CHAPTER 01

1.0 INTRODUCTION

Mushroom is the fleshy, spore-bearing fruiting body of a fungus, usually developed

above the ground on soil or at its source of food, often in wood. Perhaps it's the well

known and documented edible forest product (Zhang et al., 2014). Mushrooms were

expansively appreciated and acceptable food in the world from ancient times. In China,

Korea, Japan, Central and North American countries for their nutritional value and

medicinal properties. It has high protein content (29%) and a high content of various

types of vitamins (B, C, D, K), minerals (potassium, phosphorus), trace elements

(selenium), and low fat. Mushrooms contain a substantial amount of dietary fibers, as

well (Badalyan, 2014).

Nowadays, more than 100 countries are practicing in mushroom production. The

annual production of mushroom already crossed 5 million metric tons annually in the

world the expectation of after ten years this production reaches 7 million metric tons

(Kumar et al.,2013). Decaying cellulosic materials, deadwood parts, soil, and manure

pits these are mushrooms can grow in nature on fleshy sporophores of fungi. The

majority of these fungi belong to the class Basidiomyctoina and a few to the class

Ascomycotina (Satankar et al., 2018).

Most people have known and used wild edible mushrooms as food since time

immemorial. But this type of mushroom is seasonal, available only during the rainy

season. After that, it has been observed and studied how to cultivate mushrooms

successfully. Nowadays mushroom cultivation is developed with the advance of

science and technology. It is considered one of the important food items rich with

medicinal values since ancient times consumption of mushroom continues to increase

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for its significant role in human health and nutrition. The local demand for mushrooms

in Sri Lanka is also steadily increasing. In Sri Lanka the export development board is

located in Ratmalana, it is cultivating oyster mushroom and straw mushroom. This

board improves yields of mushrooms for local consumption and export (Gamage et

al., 2018). Mushroom cultivation is starting from mushroom spores, which grow into

mycelium and expand into a mass sufficient in volume and stored up energy to support

the final phase of the mushroom reproductive cycle, which is the formation of fruiting

bodies or mushrooms. It takes between one to three months from start to finish

depending on species (Marshall and Nair, 2009).

Many types of mushrooms have very low starch content so the diabetic patient can

take this as a good meal. Commercially there are four types of mushroom are cultivated

these are Agaricus bisporus (white button mushroom), Volvariella spp (paddy straw

mushroom), Lentinus edodes (Japanese mushroom), and Pleurotus spp (Oyster

mushroom). Normally Pleurotus type of mushroom spread all around the world in its

natural habitat, mainly in forest areas. P. ostreatus, P. pulmonaris, P. sajorcaju,

P.cornucopiae and P. ostreatoroseus are the main species of the Pleurotus genus

(Bononi et al., 1999).

Oyster mushroom, fresh fruiting body indicates a high quantity of moisture (90.8%).

After harvesting moisture will be a loss, shrinkage and rapid spoilage, so the color and

texture are changing. Depend on varieties usually mushroom shelflife duration is 2 to

5 days. So increase the self-life of the mushroom there are many methods of

preservation these are canning, freezing and drying on them in canning is the

widely used method on a commercial scale, it is quite expensive (Kulshreshtha, 2009).

Bacteria and fungi inside the mushrooms can cause spoilage during storage. Therefore,

soon after harvesting, they are either marketed or preserved with special care, such as
2
stored under a controlled environment. Processing is necessary for the mushroom's

better shelf life. They should be cooled to 0-2℃ storage temperature within five hours

of harvesting. Mushroom is kept in a packed container wrapped in plastic film at 8-10

ºC under short-term storage. There are many ways of preserving mushrooms for longer

duration such as drying, pickling and canning. Drying is done by removing enough

water to inactivate the micro-organisms (Satankar et al., 2018).

When the mushroom were dried generally color is changing to brown because during

that elevated temperature amino acid and carbohydrate are involving enzymic or non-

enzymic reaction (Kotwaliwale et al.,2007). Dehydration of mushroom is combining

hot air and microwave treatment after this type of treatment we were got a quality

product of satisfactory hydration and flavor retention (Riva et al., 1991). Dried

mushroom contains more than 25% protein and also almost 50% of total carbohydrate

with less than 3% crude fat (Kotwaliwale et al.,2007).

For the mushroom powder making, dried mushroom allows hammer mill and pass

through a 60 mesh sieve then gets the powder and pour in a low-density polyethylene

bag, sealed and store in refrigerator at 4℃ (Okafor et al., 2012). This study was carried

out to improve the nutritional and functional properties of bakery products by

supplementing wheat flour with mushroom powder. The experimentation involved

developing of mushroom powder, evaluation of the effect of wheat supplement with

this powder on rheological, physical and chemical properties of products, and

evaluation of organoleptic properties of the product with an agreeable stage of

mushroom powder addition (Majeed et al., 2019). Mushroom is consumed as baked,

fried, boiled, creamed, roasted, pickled and stuffed (Kumar et al., 2013). The

development of a value-added product such as mushroom biscuit mushroom soup

mushroom pickle, mushroom patties and pakoda from the oyster mushroom. Delicious

3
and crunchy mushroom biscuits were prepared by using the powder of oyster

mushroom and various ingredients (Wakchaure et al., 2010).

Biscuit is normally a baked food product it is called as cookie or cracker in most parts

of the world. The association of wheat consumption with such health problems as the

celiac disease makes it pertinent to utilize composite flour in biscuit manufacture. The

hardness and sweetness, the texture of the biscuits are different because of the types of

ingredients. The principal ingredients are flour, fat, sugar and water while other

ingredients include milk salt flouring agent and aerating agent. Biscuits are easy to

store, easy to carry and bring that for long journeys (Wade, 1988). The development

of a commercially viable biscuit attractive to children and adults that would have a

significant reduction in fat and sugar, with fewer calories and contain nutrients

designed to reduce the risk of coronary heart disease is highly desirable.

The biscuits are nutritive snacks produced from unpalatable dough that is transformed

into a savory product using the application of heat in an oven. They are convenient,

ready to eat and cheap food products, containing digestive and dietary principles of

vital importance (Nilugin et al., 2015). Therefore, the present study was undertaken to

develop the protein-enriched biscuits supplemented with mushroom powder as an

ingredient for the manufacture of normal biscuit.

Therefore, this research study was carried out with the following objectives;

 To develop protein-enriched biscuits using composite wheat flour and

mushroom powder.

 To find out the feasibility of partially replacing the wheat flour by mushroom

power for the biscuit making technology.

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 To study the nutritional value, microbial safety and the shelf life of the

developed biscuits.

 To assess the consumer acceptability of the product by sensory evaluation.

5
 CHAPTER 02
2.0 LITERATURE REVIEW

2.1 Biscuits

Biscuit is the most commonly produced baking commodity in the world. They are rich

in sugars, fat and calories, but poor in fiber, vitamins, and minerals rendering it unsafe

for daily usage. Longer shelf life, better taste and its position as snacks, due to its

acceptability in all age groups. This is considered a good Protein fortification drug and

other dietary enhancements (Mishra and Chandra, 2012). The bakery industry is

constantly innovating, and biscuits are products appreciated and consumed by various

categories of consumers worldwide. The development of this kind of commodity is

therefore antioxidants combined with human products that may be desirable of

customers constantly concerned about the alternative for healthy food (Caleja et al.,

2017).

The primary ingredient used to produce biscuits is wheat flour, which is low in many

nutrients including vitamins, minerals and dietary fibers (Ayo and Nkama, 2003).

Often absent in wheat flour are important amino acids including lysine, tryptophan

(Awan et al., 1991). Enrichment of biscuits with supplements like protein and vitamins

is of current interest because of the nutritional awareness of consumers (Prodhan et,

al., 2015). Wheat flour is unique because of the existence of gluten forming proteins

(glutenins and gliadins) that provide elasticity and extensibility to the wheat flour

dough, respectively (Brites et al., 2018).

Unfortunately, cookies typically produce large amounts of quickly digested starch,

sugar, and butter and small amounts of dietary fiber that nutritionists suggest make

them a very poor part of our diet. Bakers are well aware of these issues and have

6
shown some concern in them to create biscuits that can be perceived to be healthy

products but require low butter. Bakery work has so far tried to create nutritious goods

by integrating different ingredients into biscuit mixes to enhance their nutritional and

textural qualities. The key attributes of biscuits are texture, flavor, and appearance

which affect the acceptability of biscuits (Klunklin and Savage, 2018). Competition

on the market and increasing demand for safe, fresh and functional items, attempts are

being made to enhance the nutritional content and quality of biscuits by altering their

nutritional composition. Those results are most frequently accomplished by raising the

ratio of raw materials other than wheat or various forms of dietary fibers in simple

recipes to increase the protein and mineral value of biscuits for consistency and

availability or increase the fiber content of the diet and enhancing the prebiotic

characteristics of the final product (Masoodi et al., 2012).

Composite flour production refers to the method of combining various flours to allow

economical use of the local raw material to manufacture high-quality food goods.

Composite flour composition is essential to the production of value-added goods with

optimum quality (Rehman et al., 2007). Many developed countries have initiated

projects to evaluate the feasibility of alternative supplies for wheat flour replacement

of the shortage of wheat (Khader, 2001). The most severe deficiencies in children in

developing countries arising from under-nutrition are the various types of protein-

energy malnutrition (Serrem et al., 2011). Protein fortification of biscuits is of current

interest, because of increasing awareness of consumers towards health. Protein-

fortified cookies contain concentrated nutrients (Kârklina et al., 2012). Sri Lanka faces

one of the most serious nutritional problems with the shortage of protein-energy.

According to the 2006 Demographic and Health Survey, child nutrition status in Sri

Lanka reported that the incidence of malnutrition in children under 5 years of age is

7
29.4% underweight, 14% wasting and 13.5% stunting (UNICEF 2008). A dietary

protein should include all essential amino acids, in the best proportion for full body

use. Strong meat protein prices constrain the daily intake of meat protein in most

developing countries vegetable proteins provide great potential as a direct source of

protein for human consumption (Banureka and Mahendran , 2009).

Maintaining the consistency of baked goods like crackers, cookies, and biscuits were

considerable economic significance as these items are commonly consumed and are s

ometimes preserved for long periods before use (Reddy et al., 2005). In the Sri

Lankan economy, the biscuit industry plays a major role which generates foreign

exchange for the country. In this industry, the low cost of production must be

maintained to generate profits (Abayarathne et al., 2013). The per capita content of Sri

Lanka biscuits is 4 kg higher than India at 2.1 kg and Indonesia at 1.6 kg and the annual

intake of biscuits has risen to 81,000 metric tons and is worth 36 billion rupees in 2011

(Sarvananthan, 2011). Mushrooms have great potential due to their high and high-

quality proteins (20–40% on a dry weight basis), vitamins (Vitamin B- complex) and

minerals (Singh et al, 1995). Thus, mushrooms can be dried and converted into a

powdered form that can be used in baked products such as bread, biscuits, etc (Farzana

and Mohajan, 2015).

2.2 Ingredients used for Biscuit making

2.2.1 Wheat Flour

Biscuits made from wheat flour are one of the most commonly available food goods

in the world. They are an inexpensive commodity with a decent taste and shelf life

(Klunklin and Savage, 2018).

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2.2.2 Dried Oyster Mushroom Powder

Several nutritionally dense ingredients are incorporated into them for the

diversification of baking products non-wheat flours rich in protein Can be incorporated

into various products to avoid protein deficiencies. Dietary mushrooms offer a broad

range of curative properties. Oyster mushroom has important therapeutic properties

including antibacterial, antifungal, antiviral, anticancer activities, blood lipid lowering

effects and immunity. Mushrooms have also been considered helpful against insomnia,

cancer, asthma, diabetes, cholesterol reduction, allergies and stress (Wang et al.,

2010). Color changes during drying are mostly in the form of browning caused by

enzymatic or non-enzymatic reactions between carbohydrates and amino acids at

elevated temperatures (Kotwaliwale et al, 2007).

2.2.3 Sugar

Sugars like sucrose have multiple functionality in these food products, apart from

providing sweetness. Sugars have a major impact on the phase transitions of

biopolymers such as starch and gluten in bakery products. the boiling point of water,

and the sorption of water. Often, the crystalline nature of sucrose contributes

significantly to the texture of food products like biscuits, and confectionery like

fondants and creams. During their dissolution in the biscuit dough mixing stage, the

dissolving sugars compete for water with gluten. The degree of gluten hydration will

determine the spread and rise of the biscuit (Sman, 2017).

2.2.4 Palm oil

The third most essential component used in cookie-making is quantitatively fats and

oils. From the perspective of sensory consistency, fat is one of the principal ingredients

9
that influence the overall texture of cookies. Decreasing the fat content or substituting

fat with different components, as discovered in numerous earlier studies, has a huge

influence on the texture characteristics of biscuits (Zoulias et al., 2002; Rodriguez-

Garcia et al., 2013). It has been found that increasing fat levels increase the tenderness

of cookies and decreasing fat levels provide cookies with a higher breaking

strength (Lai and Lin 2006; Pareyt et al., 2010).

2.2.5 Baking powder

Baking powder is a leavening agent composed of a combination of sodium

bicarbonate, sodium aluminum phosphate, and monocalcium phosphate. The cake is

made with baking powder. Baking powder has three types are quick-moving, slow-

moving, and double-acting. Rapidly behaving baking powder reacts when shaking, late

behaving baking powder reacts when roasting, and double-acting baking powder reacts

when shaking and roasting (Ningsis, 2015).

2.2.6 Fat

Fat plays an important role by imparting shortness, richness, and tenderness, thereby

improving the eating quality of the product. The type of fat used in the preparation of

biscuits determines the quality of the end product (Jissy and Leelavathi, 2007). In the

production of biscuits, fat serves as a shortening function which makes biscuits crispier

and crumb (Lean, 2013; Gallagher et.al, 2005). keeping the cake for longevity, adding

nutritional value, giving the cake flavor and having the cake feel fluffy is the role of

fat. Fat also serves as emulsifier making a crispy cookie. There are two kinds of fat

butter or margarine, that can be used.

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2.3 Biscuit production

The traditional industrial methods of biscuit manufacturing consist of five major stages

are mixing the ingredients, biscuit forming, baking, cooling, and packing. The key

ingredients in the manufacture of shortbread cookies, which contain for example flour,

fats (e.g. butter or vegetable oils), Sugar and eggs may be combined in one or more

phases in a dough-kneading process for different period intervals and varying speeds.

Throughout this procedural step, the temperature of the dough, which is usually set in

the range between 18 and 23ºC, is critical because it affects the kneading properties of

the obtained shortbread biscuit dough. There are different kinds of oven in the food

industry suitable for baking biscuits, the heat exchange properties of which are ideal

for the product categories. Most biscuit ovens use minimal air circulation, particularly

in the first part of the baking, where heat is transmitted mainly through heated surface

radiation. In the area of bakery products, the feeding of steam (normally saturated and

not Superheated) into the baking chamber is rarely used and, in those cases, it is

intended either for heating only or for condensation on the surface of the product to

achieve a glazed (gelatinized) Surface, it not contemplating a systematic, prolonged in

time and continuous treatment with Superheated Steam to achieve baking properly.

Also, the biscuit dough can consist of a sweetening agent chosen from the category

consisting of powdered sugar, cane sugar, icing sugar, glucose syrups with varying

content of simple sugars, honey, and their mixtures (Petronio et al., 2011).

Heat impact induces a variety of physical and chemical changes in biscuits during

baking, including gas formulation denaturation and protein coagulation, starch

gelatinization, crust forming, and browning effect, water evaporation, volume

expansion, and pore structure development (Smith 1966). In the baking process, the

product becomes preheated from the original temperature to the boiling point. In the
11
second step, the evaporation happens at the product at a constant boiling temperature.

The second phase becomes similar to the constant period of the drying literature. In

the third phase, the water transfer rate from the solid depth is greater than the

evaporation rate at the surface. However water transport to the surface primarily by

diffusion as long as the continuous drying rate prevails. It is not usually preferred to

dehydrate the baked goods, the falling rate period does not always occur (Skjöldebrand

and Hallström,1980).

The baking also changes the color of the surface of the biscuits, called the browning.

There are three primary processes of browning: caramelization of carbohydrates,

dextrinization of starch and Maillard reaction in the reduction of sugar and amino

acids, in combination with the manufacture of biscuits, when the surface of the biscuit

is still dried and the temperature is above 100ºC The packaging is used as a foil to

cover the biscuit to avoid degradation after moisture is absorbed from the environment

which often avoids cranks to damage by using the appropriate packaging materials

(Whiteley, 1971).

2.4 Technique for making biscuit

The traditional industrial methods of biscuit production include five main steps:

mixing the dough's ingredients, forming the biscuits (molding process), baking,

cooling, and finally packaging (Petronio et al., 2011).

2.4.1 Mixing process

The manufacturing of biscuits, the main components, such as flour, fats (e.g. butter or

vegetable oils), sugar and eggs, may be combined in one or more phases in a dough-

kneading process with varying periods and different speeds. During this procedural

12
step, the temperature of the dough, normally set in the range between 18-23ºC, is

critical since it affects the kneading characteristics of the obtained shortbread biscuit

dough.

2.4.2 Formation of biscuit (Molding process)

Molding was intended to obtain cookies of the same shape and to increase the

attractiveness of hand molding and then cut with a knife. This invention to provide a

uniform system for preparing biscuits having substantially equal quality without

creating waste dough which would otherwise have to be reworked or discarded

(McCaffrey, 1986).

2.4.3 Baking process

Baking process is the most important factor in making cookies. The biscuits thus

shaped are then sent to the baking oven where they are subjected to a baking regime

that may differ, usually about 210°C, depending on the substance to be produced.

About 10 minutes, and more commonly between 190 and 230°C Around 8-15 minutes.

The oven should not be too hot when the material is inserted because the outside will

precocious (Petronio et al., 2011).

2.4.4 Cooling process

Upon baking, the biscuit undergoes a cooling stage at room temperature under room

temperature conditions(25ºC) and is then sent to the packing machines (Petronio et al.,

2011).

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2.4.5 Packaging

The packaging must, of course, fulfill other requirements, the chief of which is

protection. Firstly, the fragile contents must withstand considerable handling by the

packing, dispatch, and transport departments of the manufacturers; by the retailer, and

by the customer; and be in good condition when about to be eaten. The wrapper,

therefore, must protect against breakage. Secondly, it must also protect the contents

from contamination by dust, dirt, strong aromas, bacteria, and mold. Anyone of these

can arise during transit and storage and while on display at the point of sale. The

packaging should protect the contents from infestation by flies and other insects. The

packing‫׳‬s third role is to protect the contents from deterioration and spoilage,

particularly from an increase in the moisture content of the biscuits. Biscuits being

liable to problems of rancidity also, the wrapper should act as a grease barrier, for fat

which has seeped from the biscuit into the wrapper will readily turn rancid

(Whiteley,1971).

2.5 Overview of oyster mushroom (Pleurotus ostreatus)

Oyster mushrooms (Pleurotus ostreatus) are grown around the world due to their

simple cultivation techniques, the abundance of raw materials and the number of

species suitable for the climate (Kacharoo et al., 1997). The Pleurotus genus is based

on the characteristics of matting compatibility Contains a large family of about 40

recognized biological species. Edible oyster mushrooms have contributed to

tremendous demand in the last few years. Thus, now oyster mushrooms are the world's

second-largest produced mushroom (Kuforiji and Fasidi, 2009). Oyster mushroom

cultivation technique has become widespread, and substrates utilized by populations

differ from country to country and worldwide. Work on improving technologies to

14
produce improved yields is turned on concurrently. Workers have used substrate

strengthening technologies widely through Supplementation and output optimization

(Naraian, et al 2016).

Oyster mushroom production may play a significant role in the control of agricultural

waste which has become a problem of disposal (Das and Mukherjee, 2007). Oyster

mushroom should be found Cultivated in every form of cellulose material, such as

wheat, sawdust, rice hull, etc.(Hami, 1990). Studied the production of oyster

mushrooms on various wood sawdust, and noticed that P.ostreatus produced the

highest yield. Currently, sawdust is commonly used and is the chosen industrial form.

P.ostreatus has been reported to have provided optimum biological efficiency on

sawdust. Softwood sawdust such as mango and cashew are considered to be more

desirable than hardwood sawdust between the forms of sawdust (Pathmashini et

al,2009).

Mushrooms are used as flavorings and supplements for beef. The total mean value of

crude protein, crude fat, crude fiber, ash, and carbohydrates of fresh mushroom

samples was 28.85%, 2.47%, 12.87%, 9.76% and 48.16% compared to 25.91%,

2.18%, 10.41%, 10.91% and 48.16% of dried mushroom samples (Tolera and Abera,

2017). Several species of mushrooms are of great importance because of their

medicinal importance; for example, they are active against hypercholesterolemic

conditions, hypertension, diabetes, cancer and, infections. Mushroom can be a healthy

cereal complement in enriching one's diet. Mushrooms are increasing popularity in

today's healthy diet, due to their strong nutritious and fast digestibility qualities (Bobek

and Ginter, 1993).

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Sun-drying, cabinet airdrying, fluidized bed drying, freezing and Osmo-air were the

different methods involved in the dehydration of the mushroom. The characteristics of

the textural profile analyzed included firmness, firmness-resistance, cutting-force,

cutting-energy, fracture-force, and fracture-energy. For oyster mushrooms dried by the

different methods, a few consistency parameters of protein content, catalase and

peroxidase activity were examined. The quality associated with dried mushroom in

terms of protein content and residual enzyme activity is also essential for its acceptance

and preservation for further use. When dealing with very temperature-sensitive

materials, such as mushroom selecting the correct drying process may be the main

factor in the production of high quality dried goods (Arumuganathan et al., 2010). The

cultivation of edible mushrooms can be divided into two major stages. The first stage

involves the preparation of the fruiting culture, stock culture, mother spawn, and

planting spawn, while the second stage entails the preparation of the growth substrates

for mushroom cultivation.

2.5.1 Importance of Mushroom

Mushrooms with their flavor, texture, nutritional value and high productivity per unit

area have been identified as an excellent food source in developing countries to

alleviate malnutrition (Sharma et al.,2013). Mushrooms provide considerable uses in

addition to their main ecological functions, as they can be used as food and medicines.

We represent one of the worlds of the future's greatest untapped natural opportunities

and palatable products. Mushrooms were shown to be successful against cancer,

lowering cholesterol, fatigue, depression, asthma, allergies and diabetes (Bahl, 1983).

Since of high protein content, they may be used to fill the difference in protein

deficiency. Mushrooms as usable ingredients are used as vitamin replacements in the

shape of tablets to enhance immunity. These are suitable for diabetic and heart patients
16
because of reduced carbohydrate content and low cholesterol (Nanba, 1993; King,

1993).

Mushrooms are known to effectively accumulate various trace elements (metals and

metalloids such as Se or As), a phenomenon that occurs in their fruiting bodies at levels

that may significantly surpass those found in the above-ground sections of plants or

animal tissues. It is important for food and health protection that substrates used to

grow mushrooms have the lowest possible toxic elements, including As (Mleczek et,

al, 2015). The usage of mushrooms with medicinal properties is growing day by day

because of the number of side effects that traditional medicines cause. Among natural

items, mushrooms have been recognized in clinical trials as effective candidates

because they are readily available in reasonably large amounts and are cheap (Seo et,

al, 2003). Most of the world's poor live in family farms or are primarily working.

Strengthening the mushroom production sector could be essential if the rural economy

is to maintain its vitality and development, increase and diversify business and

employment opportunities in rural areas and Providing small-scale family farm income

opportunities (Sharma et al., 2013).

Production of mushrooms is constantly increasing. Commercial production in 2012,

7.959.979 tons of mushrooms were harvested, with China accounting for the majority

of the production (5.150.000 tones), while Europe harvested 1.869.091 tons (Grujic et

al., 2015). The world's population and consumption rise Mushroom demand are

forecast to grow by 15 % a year (Kamarudzaman et al., 2015). The genus Pleurotus is

a cosmopolitan group of Mushroom with high nutritional value and therapeutic

properties, in addition to a wide range of biotechnological and environmental

applications (Knop, et al., 2015). Usually considered as oyster mushrooms, these

edible basidiomycetes are among the most popular in the world, just as they have
17
achieved third place in the production of edible mushrooms, behind the species of the

Agaricus and Lentinula (Fernandes et al., 2015).

2.5.2 Taxonomy of Oyster Mushroom

Domain: Eukaryota

Kingdom: Fungi

Phylum: Basidiomycota

Subphylum: Agaricomycotina

Class: Agaricomycetes

Subclass: Agaricomycetidae

Order: Agaricales

Family: Pleurotaceae

Genus: Pleurotus

Species: P.ostreatus

Botanical name: Pleurotus ostreatus

(CABI, 2019)

The remarkable genetic diversity of the oyster mushrooms, which includes adaptation

to a broad range of environmental conditions and substrates, has placed taxonomists

in a challenging situation because it is difficult to precisely delimit Pleurotus

organisms. Conventional methods (fruit body anatomy, microscopic analyses,

population-to-people mating experiments, biochemical analyzes) did not produce

consistent findings. This has been seen that detailed molecular analyses are more

helpful: Intra- and interspecific heterogeneity was calculated through ribosomal and

mitochondrial DNA analyzes, and phylogenetic analysis of ribosomal DNA sequences

revealed regional speciation in many groups (Carrera, 1998).

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The concept of the morphological species is dominant in the field of fungal taxonomy;

most fungi are classified using morphological characteristics. However,

morphological characteristics of higher fungi are inconsistent and unstable criteria

because they are strongly influenced by the climate, cultivation substrate, and

environmental conditions. As a result, different taxonomists have formed different

conclusions regarding the taxonomic status of the same taxon based on morphological

characters. Another important taxonomic concept is that of the biological species. If

two species are inter-compatible, they are grouped as one biological species. Given the

broad acceptance of this idea, mating compatibility tests have been used to evaluate

the taxonomic identity of fungal species classified by morphological characters (Bao

et al., 2004).

2.5.3 Origin and Distribution of Pleurotus Mushroom

The origin of Pleurotus was first cultivated during the First World War in Germany as

a subsistence measure for food storages and the first documentation of cultivation was

done by Kaufer (Kaufert et al.,1936). The genus Pleurotus comprises approximately

40 species and is commonly referred to as "oyster mushroom," grows extensively in

tropical and subtropical areas and is easily cultivated artificially (Deepalakshmi and

Mirunalini, 2014).

2.5.4 Cultivation of Oyster Mushroom

After Agaricus bisporus, Pleurotus ostreatus is the second most cultivated edible

mushroom in the world. This has healing qualities and economic and ecological

qualities. With the growth of many mushrooms, the Mushroom culture has moved

towards diversification. The edible mushroom will colonize and degrade a large range

of lignocellulosic substrates and other waste that is produced primarily by the

19
agricultural, forest and food-processing industries. Specifically, P. Ostreatus takes a

shorter period to mature compared with other edible mushrooms (Sanchez, 2009).

In nature, Pleurotus ostreatus is a successful lignin degrader in the forest and it occurs

from late fall to spring in the cluster on dead trees. It is distributed worldwide,

especially in the zone of subtropical and temperate forests. Mycelial growth is a

fundamental phase which creates suitable internal conditions for cultivating

mushrooms. Thus excellent mycelium production is a very important factor in the

cultivation of mushroom (Pokhrel, 2016). Wheat straw is the main portion in European

countries for growing oyster mushroom, although adequate production can be

achieved by using a wheat straw with additional supplements that substantially

increase the yield per unit weight. The widely used substrate for cultivation of the

oyster mushroom in Asia is rice straw (Zadrazil and Grabbe, 1983; Laborde et al.,

1985; Chang et al., 1981).

Mushroom requires a fundamental understanding of its physical, mechanical,

biochemical, and enzymatic properties for the handling of Pleurotus species. The

composition of the culture media, substrates, particle size, pH, moisture, carbon to

nitrogen ratio (C/N), spawning amounts, spawning consistency, light, temperature,

humidity, and subsequent conditions involve many factors influencing the fruiting

body shape, yield and period of oyster mushroom growth (Satankar et al., 2018)

The cultivation of Pleurotus spp. has been tested in different bagging systems like

trays, cylindrical containers, wooden or polystyrene racks, blocks and plastic bags

(Quimio et al., 1990). Cultivation in plastic bags were reported to produce more

harvest than other forms with lower levels of contamination (Zadrazil and Grabbe

1983). In Europe, growers use mainly large black perforated bags while in Asian

20
countries they prefer smaller ones where inoculation and harvesting were managed at

one end of the bag (Mandeel et al., 2005).

2.5.5 Characteristics of Mushroom flour

2.5.5.1 Physical Characteristics

 Colour

One of the most important factors for assessing the consistency of flour goods is color.

Consumers would usually choose strong white wheat. The degree of white is

calculated using a color-reader. Using this method white degree of flour is

quantitatively read as L's value. The L-value has ratings varying from 0-100. The

brighter flour was measured, the L value will get closer to 100 and vice versa. The

dried oyster mushroom flour with rotary vacuum dryer 60.71 was not substantially

different from the lightness L of the dried sun-drying oyster mushroom flour 63.05.

That was reported because a temperature of 60 ºC is used for the rotary vacuum dryer

but the drying period is very low and is 8 hours. Although a temperature of 32 ºC, 6

hours a day for 4 days or in total is used with the sun drying process, the drying period

is 24 hours, meaning that both methods produce a mild browning reaction (Djamila

and Bahariwan, 2020). Adding by-products of oyster mushroom powder provides the

brownish color. It is done by the brown color of the mushroom powder such that the

concentration of the oyster mushroom powder applied to the brown color

concentration was raised (Tupamahu and Budiarso, 2017).

 Water holding capacity

Defines water holding capacity (WHC) as the strength of products to retain water

without any external force except gravity and atmospheric pressure. Oyster mushroom

21
flour dried by rotary vacuum dryer in binding water is less than sun drying. Based on

the comparative study of the contrast of the two drying methods' average water holding

capacity, it is significantly different as drying at a rotary dryer temperature of 60 ºC

allows cell elasticity to decrease such that the absorption ability of flour to water is

decreased (Djamila and Bahariawan, 2020).

 Water activity

The water activity is linked for storability to the moisture content of materials.

Storability and performance quality are affected by fluctuating water activity level in

the material. The less amount of water action on the material the greater the material's

shelf life, while the higher the water level the lower material storability would be.

Heating temperatures and strong conductivity of electricity induce low water activity.

The value of oyster mushroom water activity resulted from drying by a rotary vacuum

dryer is less than sun drying based on the statistical analysis of the comparison of the

average water activity of oyster mushroom flour dried by sun-drying and rotary

vacuum dryer machine is not significantly different (Djamila and Bahariawan, 2020).

 Bulk density

Bulk density is the mass of particles which occupy a certain volume or space. The

value of the bulk density of oyster mushroom flour dried by the sun was less while the

drying by a rotary vacuum dryer. the oyster mushroom flour resulted from the rotary

vacuum dryer was higher and significantly different from oyster mushroom flour

resulted from sun drying. This was due to the rotary dryer using 60 ℃ so that the

drying process could be faster which caused contraction stress between oyster

mushroom structures during the drying process was also faster and can affect changes

in shape and texture due to the contraction stress between oyster mushroom structures,

22
so that porous structures are damaged and constricted. As a result of shrinkage, the

flour produced has a fairly large bulk density (Djamila and Bahariawan, 2020).

2.5.5.2 Chemical Characteristics

 Nutritional Composition

Since ancient times, mushrooms have attracted human interest and the usage of

mushrooms as food is as old as human civilization. They are of great value in the

human diet since they are high in nutrition, non-starchy carbohydrates, dietary fiber,

nutrients, and vitamin-B and have little cholesterol and minimal fat content. mushroom

proteins are of excellent quality and produce ample important amino acids (Sadler,

2003). In addition to this, the bioactive functional portion Also known as β-glucan cell

wall is found in edible mushroom. β-glucan's special feature is its contribution to

healthy properties in edible mushrooms (Manzi and Pizzoferrato 2000). Alkaline ash

and high fibre content makes them suitable for consumption for those having

hyperacidity and constipation. The chemical composition of the fresh fruiting bodies

of oyster mushroom, the spent straw can be re-cycled after growing oyster mushroom.

It can be used as cattle feed and also for bio-gas production, the slurry can be used as

manure (Satankar et al., 2018). However, it can interrupted the routine schedules of

many functions such as metabolism, sleep and body temperature are disturbed which

leads to several disorders (McClung 2007). A characteristic trait of the composition of

mushrooms is the occurrence of various enzymes, which are never or only rarely found

in other organisms. The content of oxidative oxidases, fat splitting lipases, inverting

enzymes and proteolytic enzymes in mushrooms, most investigations concentrate on

the activity of polyphenoloxidases (Espin and Wichers 1999).

23
Table: 2.1 Oyster mushroom nutrition ( P. ostreatus), per 100 g

(On fresh weight basis)

Principal Nutrient value

Energy 33 Kcal
Carbohydrate 6.09 g
Protein 3.31 g
Total fat 0.41 g
Dietary fiber 2.3 g
Vitamins
Folates 38 µg
Niacin 4.956 mg
Pantothenic acid 1.294 mg
Pyridoxine (B-6) 0.110 mg
Riboflavin 0.349 mg

Thiamin 0.125 mg
Vitamin-D 29 IU mg
Electrolytes
Sodium 18 mg
Potassium 420 mg
Minerals
Calcium 3 mg
Copper 0.244 mg
Iron 1.33 mg
Magnesium 18 mg
Manganese 0.113 mg
Phosphorus 120 mg
Selenium 2.6 µg
Zinc 0.77 mg

(Source: USDA National Nutrient database)

24
  Mushroom Carbohydrates

Carbohydrates are the primary portion of dry matter for mushrooms, typically about

50-60%. The carbohydrate comprises of different monosaccharide groups, their

derivatives, and oligosaccharides (commonly known as Sugars) and both conserve and

create polysaccharides (Glucans) which are essential for the proper functioning of the

food tract (Kalac, 2012). Carbohydrates mainly found in P. ostreatus as

polysaccharides are characterized by glycogen and indigestible types such as dietary

fibers, cellulose, chitin, α- and β- glucans, and other hemicelluloses such as mannans,

Galactans, and xylans (Manzi et al ., 2001). The glucans are present with different

types of glycosidic linkages, such as branched (1→3), (1→6)-β-glucans and linear

(1→3)-α- glucans. P. ostreatus contains a specific βglucan called pleuran, which

serves as a source of antitumor polysaccharides. The composition of these

polysaccharides in the fruit bodies varies with the strains, ranging from 37 to 48 g/100g

dry fruit bodies (Synytsya et al., 2009).

 Mushroom Proteins

Mushrooms are a good source of very good quality protein especially rich in lysine

and thus supplement well the cereal-based Indian diet. The Food and Agriculture

Organization of the United Nations (FAO) recognizes mushrooms as the right source

of protein to fight protein malnutrition in the cereal-dependent developing countries

(Rai and Arumuganathan, 2008). Protein is an important component of mushroom dry

matter. Mushroom protein digestibility to be as high as 72% to 83 %. The content of

protein in mushrooms depends on the substratum composition, pileus size, harvest

time and mushroom species (Bano and Rajarathnam, 1982). In terms of the quantity

25
of crude protein, mushrooms rank below animal meat but well above most other foods

including milk (Wani et al., 2010).

 Mushroom Fats

Oyster mushrooms have rich in fiber and low in fat contents this character is highly

beneficial for heart patients (Ashraf et al., 2013). Many types of fatty acids were found

in oyster mushrooms such as lauric acid (9.94%), myristic acid (27.59%), palmitic acid

(3.36%) Hexadecadienoic (8.74%), stearic acid (20.985%), oleic acid (6.06%), linoleic

acid (21.39%), and linolenic acid (2.41%). Linoleic acid is the fatty acid considered to

be essential in the diets of humans. Linoleic acid and alpha-linolenic acid are converted

to eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid, arachidonic

acid, and cervonic acid, which are important to health. Different authors have noted

that linoleic and linolenic fatty acids account for Omega-3-fatty acid and omega-6-

fatty acid respectively. The omega fatty acids are known to play a specific role in brain

and eye development, heart development as well as normal growth development

(Majesty et al., 2018).

 Mushroom Minerals

Mushrooms are characterized by a high level of well-assimilated mineral elements.

Major mineral constituents in mushrooms are K, P, Na, Ca, Mg and elements like Cu,

Zn, Fe, Mo, Cd form minor constituents. K, P, Na, and Mg constitute about 56 to

70% of the total ash content of the mushrooms while potassium alone forms 45% of

the total ash (Bano and Rajarathnam 1982). Oyster mushrooms have the ability to

accumulate trace elements. Cofactors such as Zn, Cu, Mn, and Fe (trace micronutrient)

of various enzymes with antioxidant functions are called antioxidant micronutrients.

26
In mushrooms, several Se compounds have been identified which act more efficiently

as an antioxidant in the form of selenoproteins that reduce the cytotoxic effects of

reactive oxygen species (Gupta et al ., 2017).

 Mushroom Vitamins

Mushrooms could be a good source of vitamins B1, B2, niacin, biotin and vitamin C.

The vitamin content is of great value since they have important functions in the human

and animal organisms (Breene, 1990). However, vitamin contents were species-

dependent. The riboflavin contents in mushrooms were higher than those generally

found in vegetables, and in A. bisporus varieties the contents were as high as those

found in eggs and cheese. All the mushrooms analyzed were rich in niacin. P.ostreatus

contained higher levels of niacin (65 mg/100 g in dry weight) than other cultivars.

Mushrooms contained moderately high amounts of folates and the contents were of

the same magnitude as that generally found in vegetables (Mattila et al., 2001).

 Browning effect

Color changes during drying are mostly in the form of browning caused by enzymatic

or non-enzymatic reactions between carbohydrates and amino acids at elevated

temperatures (Kotwaliwale et al., 2007). Mushroom tissue is a good source of both

oxidative enzymes and phenolics. Current conventional techniques to avoid browning

include autoclaved and blanching methods. The shelf life of minimally processed

mushrooms, such as Agaricus bisporus, is limited to a few days, because of enzymatic

browning during storage. Inactivation of polyphenol oxidase (PPO) in mushroom (the

principal enzyme responsible for the browning reactions) by heat or the application of

antioxidants or enzyme inhibitors is essential to prevent enzymatic browning and

27
extend the shelf life of mushroom for commercial use (Luo and Canovas 1996). The

lightest colored dried mushrooms were produced at 40°C drying temperature probably

because they undergo the minimum non-enzymic browning (Mohamed and Hoo

1994).

2.6 Packaging and Shelf-life Products

Food packaging is defined as a coordinated system of preparing food for transport,

distribution, storage, retailing, and end-use to satisfy the ultimate consumer with

optimal cost (Coles et al., 2003). Active and intelligent packaging systems can provide

multiple benefits for food quality and health. The active systems aim to prolong the

shelf-life of consumer items by preserving their quality for longer (e.g. oxygen

absorbers in nuts to avoid rancidity caused by fat oxidation). Some new developments

in active packaging have included preservative-releasing packaging systems intended

to improve the shelf-life of processed food and thereby preserve high quality (Jong et

al., 2005). Food packaging is an essential part of modern society; commercially

processed food could not be handled and distributed safely and efficiently without

packaging. The World Packaging Organization (WPO) estimates that more than 25%

of food is wasted because of poor packaging (WPO, 2009).

2.6.1 Types of Packaging Materials

Polyethylene, polymerized from ethylene, is the most widely used material for

packaging foods. In general, polyethylene has durability, good moisture control, high

oil and chemical resistance, and strong impact strength. Polyethylene is also a cheap

material, and this material is generally the most economical option for applications

where its performance is suitable (Shin and Selke 2014).

28
2.6.1.1 Polyvinyl Chloride

Polyvinyl chloride is produced from vinyl chloride monomers. PVC has high

toughness and strength, good dimensional stability, good clarity, excellent oil barrier

properties, and good heat sealing ability. Even though it has many beneficial

properties, PVC is easily degraded at high temperatures. It decomposes and gives off

hydrogen chloride (HCl) around its melting temperature. Thus, unmodified PVC is

almost impossible to process due to thermal degradation (Shin and Selke 2014).

2.6.1.2 Paper and paper board

Paper and paper board are the most commonly used packaging materials in the world.

In the US, over 50% of all packaging is paper-based, including food packaging. Paper

is produced from plant fibers. More than 95% of paper is made from wood, and the

remaining sources are mainly agricultural by-products, such as straw (of wheat, rye,

barley, and rice), sugar cane bagasse, cotton, flax, bamboo and corn husks (Shin and

Selke 2014).

2.6.1.3 Glass

Glass is defined as “an amorphous inorganic product of fusion that has been cooled to

a rigid condition without crystallizing” (ASTM, 2003). For food packaging, bottles or

jars are the types of glass packaging most often used, bottles being the primary use. In

the US, 75% of all glass food containers are bottles. Glass is made primarily of silica,

derived from sand or sandstone. For most glass, silica is combined with other raw

materials in various proportions. For example, soda-lime glass, the glass typically used

for food packaging, contains silica (68–73%), limestone (10–13%), soda ash (12–

15%), and alumina (1.5–2%). Glass is inert to a wide variety of food and non-food

29
products, very rigid and strong against pressure, transparent, and nonpermeable

(excellent barrier properties) (Shin and Selke 2014).

2.6.1.4 Aluminium Foil

Aluminium foil is the most commonly produced metal foil. It is manufactured by

passing aluminum sheet between a series of rollers under pressure. Pure aluminum

(purity >99.4%) is passed through rollers to reduce the thickness to less than 150μm

and then annealed to provide dead-folding properties. Foil is widely used for wraps (9

μm), bottle caps (50 μm), and trays for ready-to-eat meals (50–100 μm). Aluminum

foil has excellent barrier properties against gases and water vapor. Thus, it is also used

as the barrier material in laminated films for packages, such as those in retort pouches

(Shin and Selke 2014).

2.7 Sensory Evaluation

2.7.1 Definition

The sensory evaluation has been defined as a scientific method used to evoke, measure,

analyze, and interpret those responses to products as perceived through the senses of

sight, smell, touch, taste, and hearing (Stone and Sidel, 2004). The sensory evaluation

comprises a set of techniques for accurate measurement of human responses to foods

and minimizes the potentially biasing effects of brand identity and other information

influence on consumer perception (Lawless and Heymann, 2010).

30
2.7.2 Uses

 Analyze food sample for improvements

 Explore specific characteristics of an ingredient or dish/food product

 Evaluate a range of existing dishes/food products

 Gauge responses to a dish/product, e.g. Acceptable versus unacceptable

 Check whether a final dish/food product meets its original specification

 Provide objective and subjective feedback data to enable informed

decisions to be made

 Compare similarities/differences in a range of dishes/products

2.7.3. Methodology

To derive maximum benefits out of sensory evaluation, it is necessary to follow the

methodology in its full scientific perspective. The basic steps to perform the sensory

analysis are;

 selection of the proper panel

 maintaining suitable environmental conditions and use of standard equipment

for the test

 obtaining representative samples

 preparation and presentation of samples for evaluation in a manner that

 ensure the uniformity and representation of the samples

 selection of the proper methods and statistical techniques

2.7.3.1 selection of the proper panel

Sensory evaluation is normally carried out by designed experiments under proper

environmental conditions by both trained and untrained panels. The experts who have

31
acquired the product-specific skills are not appropriate for the general evaluation

because of the risk of being biased. Panels with different degrees of training are

required for the different types of sensory analysis. The degree of training required

depends on several considerations, such as the degree of differences to be detected, the

number of panelists required for the tests and time and the value of the analysis to the

product type. Training is also necessary for descriptive and profile panels using

specific procedures for specific products and situations. For my sensory evaluation

test, at least twenty members should be selected in a panel.

2.7.3.2 Maintaining suitable environmental conditions and use of standard

equipment for the test

Environmental factors, where the sensory evaluation is to be done and samples that

need to be evaluated, should be suitably controlled. sensory evaluation should be

conducted in quiet and well- lit rooms; free from any odours. The rooms should be

constructed to have comfort for concentrated prolonged testing and case of cleaning.

Pleasing neutral shades and maintenance of comfortable temperature and humidity

conditions of the whole area or at least the room where panel members are going to sit

and discuss are desirable and appealing. All these are essential to help the panel

members develop an interest in carrying out the test. The testing area where booths are

located should be separated from sample preparation and washrooms.

2.7.3.3 Sample preparation

Samples should be prepared in a way to bring out the differences in a particular quality

attribute under evaluation. All variables like temperature, time of boiling, quantity and

composition of water, blending, etc., should be controlled to ensure identical methods

32
of preparation for all samples. Care should be taken that no loss of flavors occurs and

no foreign tastes or odors are imparted by the procedure during preparation, storage,

serving, etc.

2.8 Hedonic rating test

The hedonic scale is assumed to ask each tester to taste each sample in turn and tick a

box, from dislike very much to like very much to indicate their preference. Samples

were randomly served to the panelists and asked to make remarks about the products՚

appearance, taste, odor and texture. The result should be analyzed by the panelists that

which sample received the highest or lowest score and which sample was preferred.

Hedonic rating is converted to score and treated by rank analysis or analysis of

variance. The rating labels obtained on a hedonic scale may be affected by many

factors other than the quality of the test samples.

2.9 Problems of sensory evaluation

The elevator may be biased. If the judgment on elevators may be biased, the test result

could be erroneous. Erratic information is also a problem generally informative

training sessions were not designed to respond to simple systemic analytical

questionnaires. In the absence of established and standard language, the analysis of

comments is dangerous and without the assessor's formal guidance, attention is paid

to certain issues, not all of them and the result is therefore incomplete (Lyon et al.,

2012).

2.10 Qualities assessed by sensory test

The major aspects of food acceptance are color, taste, aroma, texture and overall

acceptance.
33
2.10.1 Colour

Food surface color is the first consistency attribute measured by customers and is

important for product acceptance. Food appearance often defined by surface color is

the first sensation that the user perceives and uses as a method for embracing or

rejecting food (Leon et al., 2006).

2.10.2 Aroma

Among various factors related to food palatability, taste and aroma are the most

important factors because they directly affect the flavor perception in the mouth. The

aroma is felt by the sense of smell and also contributes to our evaluation of food

quality.

2.10.3 Taste

Taste is the most important factor in a person‫׳‬s selection of a particular food. In

humans, the taste is divided into five modalities: sweet, spicy, salty, sour and umami

(glutamate or amino acid the taste). In general, tastes of sweet, umami, and low-salt

evoke behavior approval of food whereas tastes of salty, sour, and high-salt elicit

avoidance. Although discrimination has not been studied thoroughly, mammals are not

considered to be able to distinguish numerous chemical signals in a modality (Breslin

et al., 1996; Spector and Kopka, 2002). Individuals differ in their sensitivity to the four

taste sensation and the threshold for each the primary tastes is usually not at the same

level in any one individual.

34
2.10.4 Texture

The texture is characterized as coarse or fine. Coarse textured crystalline products are

said to be granny. Major aspects of texture are considered as crisp, crumbly, tender

and hard for baked products. Texture refers to those qualities of a food that can be felt

with the fingers, tongue, palate, or teeth.

2.10.5 Overall acceptability

The most important organoleptic property of foodstuffs is overall acceptability. This

is a combination of all other sensory attributes and the assessment of this property is

difficult and differs to a higher degree than the other characteristics between persons.

35
 CHAPTER 03

3.0 MATERIALS AND METHODS

An experimental study was carried out to developed and assess the mushroom flour

incorporated Biscuit in the Food Science and Technology Laboratory of the

Department of Agricultural Chemistry, Faculty of Agriculture, Eastern University, Sri

Lanka from the period of July to October 2020. The methodology adopted has been

described below.

3.1 Material used for this study

Oyster Mushroom Baking powder

Wheat Flour Margarine

Sugar Salt

Palm oil

3.1.1 Procurement of Materials

Fresh and Good qualities of Oyster mushroom without any spoilage were procured

from locally from the Kiran area in the Batticaloa district. other major ingredients such

as wheat, sugar, salt, baking powder and margarine were purchased from the leading

supermarket in the Batticaloa district.

3.1.2 Preparation of mushroom powder

The fresh mushroom was washed and tore into thin slices by hand at around 2-3 mm

thickness manually. Drying of mushroom slices was done on aluminum trays in the

sunlight until the pieces were starting brown and then stored in a tight container till

36
further use. The dried pieces were milled into flour using an electric grinder and poured

through 80 mesh sieves in obtain flour of uniform. the flour was packed in an airtight

glass container and store under refrigerator condition until further use.

Wash the fresh mushroom cap and stalk without any dirts

Tearing cap and stalk of mushroom by using hand

Sun-drying (for 24 hours)

Milling

Sieving the mushroom powder

Packaging in an airtight container

Sealing

Storage (at 8ºC)

Figure 3.1 Flow chart for the preparation of Mushroom powder

3.1.3 Preparation of wheat flour

Purchased wheat flour was sieved to remove foreign particles and to obtain uniform

particles.

3.2 Development of Biscuits

Biscuit dough was prepared according to the following formula: 100 g of flour (contain

a different proportion of Mushroom powder and Wheat flour), and the equal amount

of margarine, Sugar (Powder form), Baking powder, Palm oil, sodium chloride salt

and various proportion of water to make required consistency of dough.

37
Biscuits were prepared by using the creaming method. The sugar and margarine were

mixed by a beater to produce a creamy mixture. Dough that contained the flour, water,

the baking powder and sodium chloride were added to the resulting cream and mixed

to obtain a homogeneous mixture. Finally, Biscuit dough had to contain the various

proportion of Mushroom powder. The firm dough was prepared from every mixture

(kind of treatments). The dough was rolled out to 5mm thickness in a baking tray and

cut into a round having 5.0 cm diameter with a biscuit cutter.

The biscuits were placed on a greased aluminum tray and baked in a preheated oven

at 180 ºC for 20 minutes to produced Biscuits. The baked biscuits were cooled and

assessed for sensory analysis.

Preparation of composite flour (Mushroom powder: wheat flour)

(0:100, 5:95, 10:90, 15:85, 20:80)

Premixing the margarine and sugar (cream)

Mixing the flour, baking powder and salt with cream

Sheeting (5mm thickness)

Baking (at 180 ºC, 20 min)

Cooling

Packaging in airtight containers

Storage (30ºC)

Figure 3.2 Flow chart for the preparation of Biscuits

38
3.3 Different combinations of Mushroom powder and wheat flour for composite

Biscuits. (Experimental Design)

Table 3.1 Experiment design

Treatment Mushroom powder (%) Wheat flour (%)

T1 (234) - 100%

T2 (452) 5% 95%

T3 (326) 10% 90%

T4 (425) 15% 85%

T5 (212) 20% 80%

Table 3.2 Treatments of preparation of Mushroom Biscuits

Treatmen Wheat Mushroom White Margarine Palm Salt Baking

ts flour (g) flour (g) sugar (g) oil (g) (g) powder
(g) (g)

T1 100 - 30 35 15 1 0.5

(Control)

T2 95 5 30 35 15 1 0.5

T3 90 10 30 35 15 1 0.5

T4 85 15 30 35 15 1 0.5

T5 80 20 30 35 15 1 0.5

3.4 Organoleptic Analysis

Twenty trained panels of judges carried out the organoleptic evaluation of the above

five different biscuit samples. The panel consisted of some students, staff members

39
and other persons. each judge was given a sample of each treatment for the evaluation.

All five different Biscuits were named as treatment. The quality factors such as colour,

taste, flavor, and overall acceptability were measured by a seven-point hedonic

structure scale. Scoring scales as follows,

7 = Very much like

6 = Much like
5 = Like
4 = Neither like or dislike
3 = Dislike
2 = Much dislike
1 = Very much dislike

Plate 3.1: Biscuits prepared with different amounts of added Mushroom powder

A questionnaire that contained all the details about the sensory evaluation was given

to each panelist. This evaluation was conducted from 9.00 am to 12.30 pm of the day.

Each sample was arranged randomly for the evaluation.

3.4.1 Materials used for Organoleptic analysis

The biscuits of each treatment Questionnaire list

Mineral water

Pen

Disposable dishes

Paper tissue
40
 Plate 3.2: Arrangement of Biscuit Sample for Organoleptic analysis

3.4.2 Serving the samples for Organoleptic analysis

Samples were kept in disposable plates separately. Then plates were labeled according

to the coding numbers. The order of the samples was randomized when those serving

to the panelist. One biscuit was served from every treatment to the panelist. The

following instructions were given to the panelist before doing the sensory evaluation.

 Should consider the taste, color, texture, flavor, and overall acceptability of the

biscuit product.

 Should indicate the scale numbers for all samples

 Should give an order of preference

 Should not communicate with other panelists during sensory evaluation

3.5 Physical properties of wheat flour and mushroom powder composite biscuits

Wheat flour and mushroom powder biscuits were measured and calculated for its

physical parameters such as diameter, thickness, volume, density, and spread ratio

3.5.1 Diameter

The diameter of biscuits was measured by using Vernier caliper.

41
3.5.2 Thickness

The thickness of the biscuits was measured by a ruler. Five biscuits were placed one

on another and the total height of all five biscuits were measured by a ruler. This

process repeated thrice and the average measurement was taken as reading

3.5.3 Volume

Volume of the biscuits was calculated by the area of the biscuit was multiplying its

thickness

3
3.14 𝐻𝐷2
Volume ( cm ) =
4

Where,

H = Thickness of biscuit (cm)

D = Diameter of biscuit (cm)

3.5.4 Density

The density of biscuits was obtained by the ratio of mass to volume.

Mass of biscuit ( g )
Density (gcm−3) =
Volume of biscuit (cm3 )

3.5.5 Spread Ratio

The spread ratio is determined by the ratio of diameter into the thickness of biscuit.

Diameter of biscuit ( cm)

Spread ratio =
Thickness of biscuit ( cm )

42
3.6 Nutritional Analysis

Biscuit selected from the sensory evaluation were analyzed for nutritional qualities

such as moisture, ash, protein, fat, fiber by proximate analysis according to AOAC

(2002) methods. The parameters were analyzed initially after formulation and during

the storage period. Analyses were carried out for three treatments which are good

samples selected from the sensory evaluation.

3.6.1 Determination of Moisture content

Principle:

The moisture content is determined by oven drying the food product to a constant

weight at 100 – 105℃ for overnight. Then loss in weight of the food product is mainly

moisture.

Materials

Moisture canes

Oven

Electric balance

Biscuit samples of different treatments

Procedure

The weight of empty moisture can was taken. 5g of each sample were measured and

these samples were added to moisture cans. The weight of moisture cans with sample

was measured. Moisture cans were placed in the oven for overnight at 105- 110 ºC

temperature. After drying, moisture cans were cooled in desiccator and oven-dry

weight was taken

43
Calculation

The weight of empty moisture cane = M1

The weight of moisture can with sample = M2

Oven dry weight = M3

M2−M1
Moisture content = *100%
M2−M1

Plate 3.3: Oven dried Biscuits of each treatment

3.6.2 Determination of Ash content

Principle:

The ash content of the food is determined by burning the food products at 450 ºC until

all the carbon has been burnt. This fraction includes essential and non-essential mineral

elements.

Materials

Muffle furnace Desiccator

Crucible Biscuit samples of different treatment

Electric balance

44
Procedure:

The weight of the empty crucible was taken. 1 g of biscuit sample was taken to the

weighted crucible. The weight of crucible with the sample was taken. It was ignited in

the muffle furnace at 450 ºC – 550 ºC for 6 hours. Then the weight of ash was taken.

Weight of empty crucible = M1

Weight of crucible with sample = M2

Ignited weight of crucible = M3

M3−M1
Ash content ( % ) = *100 %
M2−M1

Plate 3.4: Ash of Biscuits in each treatment

3.6.3 Determination of crude Fiber content

Principle:

Fiber is a mixture of substances that make the framework of plants and composed of

cellulose, hemicellulose, pectin and lignin of cell walls. The estimation is based on

treating the moisture and fat-free material with 1.25% diluted acid then with 1.25%

alkali, thus imitating the gastric and intestinal action in the process of digestion. The

material left undissolved is considered as fiber content.

45
Materials:

1.25% NaOH Oven

1.25% H2SO4 Muffle furnace

1% HNO3 Muslin cloth

Funnel Beakers

Watch glasses

Procedure:

Biscuit sample of 5g was taken to a 500ml beaker. A volume of 200 ml 1.25% of

H2SO4 was added to it and covered with a watch glass. It was boiled for 30 minutes.

The solution was filtered through a muslin cloth and washed by hot water until the

residue-free from acid. The residue was transferred to a beaker. A volume of 200 ml

1.25% of NaOH was added to it and covered with a watch glass. It was boiled for 30

minutes. The solution was filtered through a muslin cloth and washed by hot water

until the residue-free from alkali. Then the residue was washed with 1% HNO3. It was

transferred to a previously weighed crucible and dried in oven at 105℃ until reaching

constant weight. Then the dried residue was ignited in a muffle furnace at 450-500℃.

after ignition the samples were allowed to cool in a desiccator and the weight of

crucible with ash was taken.

Calculation

Weight of crucible = M1

Weight of oven-dried sample = M2

Weight of crucible with ash = M3

46
 (M2−M1)−(M3−M1)
Fiber content = *100%
Weight of sample

Plate 3.5: Solution filtering through Plate 3.6: The Ash of burned residue
Muslin Cloth

3.6.4 Determination of crude protein

Principle:

The protein content is calculated from the nitrogen content of the food, determined by

the Kjeldahl digestion method. When the food sample is digested with concentrate

sulphuric acid the organic form of nitrogen is converted into ammonium sulphate. The

digested material containing ammonium sulphate is distilled with an excess of alkali

and the ammonium liberated is absorbed in a known excess of standard acid using

methyl red as an indicator. The unreacted acid is then determined by back titration

with standard alkali.

Procedure

A. Digestion

Biscuit sample of 2.5g was added to a Kjeldahl flask. A volume of 50 ml H2SO4, 10 g

K2SO4 and 1 g CuSO4 were added to the Kjeldahl flask. It was allowed to digest for 3-

4 hours in the digestion chamber until to get a clear solution. After the digestion flask

was removed and allowed to cool for one hour.

47
B. Distillation

In the distillation process, 0.3 g of Devardas alloy and some porcelain pieces were

added to the flask to avoid frothing. The Kjeldahl flask and a conical flask were

connected in distillation until. 200 ml of distilled water and 90ml of 40% NaOH were

added into the Kjeldahl flask and boric acid was added to the conical flask by

automatically. Then it was allowed to distillation for two minutes. After all the NH3

traped by boric acid, the conical flask was removed from the distillation unit.

C. Titration

The NH3 in the conical flask was titrated against 0.1 N H2SO4. Before titration 2 drops

of Bromo red-green indicator (mixed indicator) was added to the conical flask. The

color was changed to green. Then it was titrated against 0.1 N H2SO4 solution until the

color become pink. The burette reading was recorded.

Calculation

Burette reading = Y ml
(1000 ml 1 N H2SO4 react with 14 g Nitrogen)
14∗Y∗0.1 N
Nitrogen content = * 100%
1000∗1 N∗2

Protein content = Nitrogen content * 6.25%

48
Plate 3.7: Digestion and Distillation Units

Plate 3.8: Titration unit Plate 3.9: Endpoint of titration

3.6.5 Determination of fat

Principle :

The fat extracted from the moisture free material with petroleum ether of boiling point

40 - 60 ℃. The ether extract is determined by subjecting food to continuous extraction

with petroleum ether for 3 – 4 hours or more. The residue after evaporation is ether

extract content.

Materials:

Soxhlet extractor Petroleum ether

49
Thimble Filter paper

Electric balance Oven

Water bath

Procedure:

The extraction flask was washed and dried in oven at 105℃ and cooled down in

desiccators and the flask was weighed. A sample of 2.5 g was weight and transferred

to an extraction thimble. The thimble was then transferred into the Soxhlet extractor.

Around 300 ml of petroleum ether was taken into extracting flask and kept on the

heating mantle. Soxhlet was connected, the water was allowed to turn to cool them and

the heating mantle was switched on (40 – 60℃). They were allowed to distillation for

6 hours. After complete of distillation, the thimble was removed from Soxhlet

extraction. Then the flask was kept in a water bath for 10 minutes to evaporate the

excess ether. After that, the flask was kept on over at 70℃ to remove the water and

the constant weight of the flask was weighed.

Calculation:

Weight of empty Soxhlet flask = M1

Oven dry weight of empty Soxhlet flask = M2

Weight of food sample = M3

M2−M1
Fat (%) = *100%
M3

50
Plate 3:10: Fixed the extraction Plate 3:11 Extracted fat

3.7 Microbiological Analysis

Materials

Potato – 200 g Distilled water - 1000 ml

Sucrose – 20 g Agar – 30 g

Procedure

The potato was peeled, cut into small pieces and added in 250 ml of water and boiled.

Agar was placed in a 1000 ml flask with 250 ml of water and boiled until agar

dissolves. The required amount of sucrose and potato extract was added into the flask

and stirred. The conical flask containing media was closed with cotton wool and

wrapped in aluminum foil. The conical flask was put into the autoclave at 121℃, 15

psi for 20 minutes and the media was allowed cool.

Plate 3.12: Sterilized PDA media

51
3.7.1 Total plate count

Materials:

The PDA media Petri dishes

Alcohols Spirit lamp

Cotton wool with Aluminium foil

Procedure:

Petri dishes, forceps and needles were kept in the oven at 180℃ for one hour and

allowed to cool. All the types of equipment were sterilized by 70% of alcohol. Then

was poured into the petri dishes and they were kept in the laminar flow until solidify.

The different treatment samples were placed on the agar plate. Then petri dishes were

covered and labeled. The plates were observed after 4 days for plate count.

Plate 3.13: Petri dishes with media and biscuit sample of each treatment

3.8 The storage studies

Based on the physical, nutritional and sensory analysis most preferred biscuits samples

were selected for storage studies. Three replications were prepared for each treatment

for the evaluation three kinds of treatments were selected for storage study these are

52
T1, T3 and T4. These biscuits were packed in sealed laminate aluminum foil which is

commercially used to pack the biscuits. Biscuits packs were store under the ambient

condition of the average temperature of 30℃ and relative humidity 75% - 80% for 2

months.

Plate 3.14 Storage analysis of Mushroom biscuit

3.9 Shelf life evaluation

The shelf life of biscuit was assessed based on the nutritional, sensory, and

microbiological qualities. The nutritional qualities of each treatment were tested at 2

weeks interval for two months by proximate analysis. A microbiological test was

carried out by total plate count to assess the safety of the biscuit samples. Sensory

analysis was carried out at the end of two months.

3.10 Statistical analysis

Data of the chemical analysis and storage study were analyzed by Analysis of Variance

(ANOVA) (α =0.05) and mean separation was done with Duncan's Multiple Range

Test (DMRT). Data related to sensory evaluation were analyzed using Turkey's Test.

Both chemical and organoleptic analysis was done through statistical analysis system

(SAS) software statistical package.

53
 CHAPTER 04

RESULT AND DISCUSSION

4.1 NUTRITIONAL COMPOSITION OF THE FRESHLY MADE OYSTER

MUSHROOM POWDER

The nutritional composition of the freshly prepared oyster mushroom powder is

present in the moisture content of oyster mushroom powder was 10.04%. Oyster

mushroom powder had a fiber content of 12.18%. the ash content of oyster mushroom

had 4.79% and the protein content of mushroom powder was 28.33% results of the

nutritional composition of mushroom powder are closely related with the results

obtained by Djamila and Bahariawan (2020). The fat content of oyster mushroom

1.90% of this fat content is also closely related to the result obtained by Okafor et al.,

(2012).

4.2 PHYSICOCHEMICAL ANALYSIS OF MUSHROOM BISCUIT

4.2.1. Physical Characteristics of developed mushroom biscuits

The physical parametric analysis of the biscuits revealed, there were significant

differences between the treatments as the level of mushroom powder was increased

(0-20%) for diameter, thickness, volume, density, and spread ratio of biscuits at the

5% level of significance according to ANOVA.

4.2.1.1 Diameter

There was a significant decrease in the diameter of the content of control treatment

(T1) and other different treatments (T2, T3, T4, T5) after incorporating biscuits with

mushroom powder, According to DMRT, control treatment (T1) which containing

54
100% of wheat flour showed significantly (p < 0.05)has highest mean value (5.02 ±

0.01) and T5 treatment contain 20% mushroom powder and 80% of wheat flour showed

significantly (p< 0.05) lowest value (4.62 ± 0.01). There was a significant difference

between the treatments.

Table 4.1: Physical Properties of freshly made of wheat flour– mushroom powder

biscuit
Treatment Density Volume Thickness
(gcm-3) (cm-3) (cm)
T1 0.73 ± 0.01e 10.60 ±0.09e 0.54 ± 0.01e

T2 0.83 ± 0.01d 12.28 ± 0.22d 0.65 ± 0.01d

T3 0.92 ± 0.01c 13.29 ± 0.07c 0.73 ± 0.01c

T4 0.97 ± 0.01b 15.18 ± 0.17b 0.87 ± 0.01b

T5 1.05 ± 0.01a 16.19 ± 0.13a 0.97 ± 0.01a

The values are the means of four triplicates ± Standard Error.

The means with the small letters are not significantly different from each other at a 5% level
based on DMRT.

4.2.1.2 Density

There was a significant increase in the density of content of control treatment (T1) and

other different treatments (T2, T3, T4, T5) after incorporating biscuits with mushroom

powder, According to DMRT, the treatment (T5) which containing 80% of wheat flour

and 20% mushroom powder showed significantly (p < 0.05)has highest mean value

(1.05 ± 0.01) and T1 treatment contain 100% of wheat flour showed significantly (p<

0.05) lowest value (0.73 ± 0.01) There was a significant difference among the

treatments.

55
4.2.1.3 Volume

There was a significant increase in the volume of content of control treatment (T1) and

other different treatments (T2, T3, T4, T5) after incorporating biscuits with mushroom

powder, According to DMRT, the treatment (T5) which containing 80% of wheat flour

and 20% mushroom powder showed significantly (p < 0.05)has highest mean value

(16.19 ± 0.13) and T1 treatment contain 100% of wheat flour showed significantly (p<

0.05) lowest value (10.60 ±0.09) There was a significant difference among the

treatments.

4.2.1.4 Thickness

There was a significant increase in the thickness of content of control treatment (T1)

and other different treatments (T2, T3, T4, T5) after incorporating biscuits with

mushroom powder, According to DMRT the treatment (T5) which contains 80% of

wheat flour and 20% mushroom powder showed significantly (p < 0.05)has highest

mean value (0.97 ± 0.01) and T1 treatment contain 100% of wheat flour showed

significantly (p< 0.05) lowest value (0.54 ± 0.01) There was a significant difference

among the treatments

4.2.1.5 Spread Ratio

There was a significant decrease in the diameter of the content of control treatment

(T1) and other different treatments (T2, T3, T4, T5) after incorporating biscuits with

mushroom powder, According to DMRT control treatment (T1) which containing

100% of wheat flour showed significantly (p < 0.05) has highest mean value (9.40 ±

0.14) and T5 treatment contain 20% mushroom powder and 80% of wheat flour showed

significantly (p< 0.05) lowest value (4.77 ± 0.03). There was a significant difference

between the treatments.

56
 12

10 9.4
Diameter (cm) and Spread ratio

8 7.65

6.66

6 5.45
5.02 4.92 4.83 4.72 4.62 4.77
Diameter
4

Spread ratio
2

0
T1 T2 T3 T4 T5
Treatments

Figure 4.1: Diameter and spread ratio of a different combination of wheat flour
and mushroom powder biscuits
The values are means of four replications.
The vertical bars indicate standard errors.

4.3 Organoleptic Qualities Analysis of freshly made mushroom biscuits

The organoleptic qualities analysis of biscuits revealed that there was a significant

difference between the treatments as the level of mushroom powder increased from 0

to 20% for texture, flavor, taste, color and overall acceptability at a 5% level of

significance according to ANOVA. Mean values of treatments according to Tukey's

studentized Range (HSD) test are shown in Table 4.2

57
Table 4.2: Organoleptic Analysis of Freshly Made Mushroom Biscuits

Overall
Treatment Color Texture Flavor Taste acceptability

T1 4.40 ± 0.23b 4.55 ± 0.19b 4.40±0.19bc 4.55 ± 0.33b 4.95 ± 0.17bc

T2 4.25 ± 0.21b 4.00 ± 0.28b 4.05±0.19c 4.50 ± 0.19b 4.45 ± 0.20c

T3 4.45 ± 0.14b 4.20 ± 0.19b 4.80±0.16b 4.75 ± 0.14b 5.25 ± 0.16b

T4 5.20 ± 0.17a 5.55 ± 0.18a 5.65±0.17a 6.15 ± 0.13a 6.40 ± 0.11a

T5 4.00 ± 0.17b 4.20 ± 0.15b 4.35±0.18bc 4.50 ± 0.14b 4.90 ± 0.12bc

Pr > F < .0001

The values are the means of triplicates ± Standard Error.
The means with the small letters are not significantly different from each other at a 5% level
based on Tukey's test. Sensory parameters were measured using a seven-point hedonic scale

The findings of the sensory attributes of mushroom powder and wheat flour blend

biscuits such as scores of color, texture, taste, flavor, and overall acceptability are in

the deviated pattern and also score of mouth feel attribute is in the similar way of

pattern compared with the result obtained by Sukhcharn et al. (2008). These different

directions of score pattern may be due to the different rates of preference and

acceptable values of panels and the quality of finished biscuits that were studied.

4.3.1 Texture

Texture analysis involves measuring the properties related to how a food feels in our

mouth (initial bite). According to DMRT, there was no significant difference between

the control treatment, 5%,10%, and 20% mushroom powder added biscuits (T2, T3, T4,

T5) but the 15% mushroom powder added biscuit (T4) showed significantly (p < 0.05)

highest mean value (5.55 ± 0.18 ) and 20% mushroom powder added biscuit (T2) has

the least mean value (4.00 ± 0.28).

58
4.3.2 Colour

According to DMRT, there was no significant difference between the control

treatment, 5%,10% and 20% mushroom powder added biscuits ( T1, T2, T3, T5) but the

15% mushroom powder added biscuit (T4) showed significantly (p < 0.05) highest

mean value (5.20 ± 0.17) and 20% mushroom powder added biscuit (T5) has the least

mean value (4.25 ± 0.21).

4.3.3 Flavour

According to DMRT, 15% mushroom powder added biscuit (T4) showed significantly

(p < 0.05) highest mean value (5.65 ± 0.17) and 20% mushroom powder added biscuit

(T2) has the least mean value (4.05± 0.19). There was no significant difference among

T1, T3 and T5 on color.

4.3.4 Taste

According to DMRT, 15% mushroom powder added biscuit (T4) showed significantly

(p < 0.05) highest mean value (6.15 ± 0.13) and 20% mushroom powder added biscuit

(T2) has the least mean value (4.50 ± 0.19). there was no significant difference between

the control treatment, 5%,10% and 20% mushroom powder added biscuits ( T1, T2, T3,

T5).

4.3.5 Overall acceptability

According to DMRT, 15% mushroom powder added biscuit (T4) showed significantly

(p < 0.05) highest mean value (6.40 ± 0.11) and 20% mushroom powder added

biscuit(T5) has the least mean value (4.45 ± 0.20). there was no significant difference

among T1, T3 and T5 on overall acceptability.

59
 4.4 Nutritional Analysis of Freshly Made Mushroom Biscuits

The result of the proximate analysis of biscuits made from wheat flour and mushroom

powder blends were analyzed. There was an increase in protein, ash, fat and fiber

contents of the composite cookies. There was decrease in moisture content of the

composite biscuits.

Table 4.3: Moisture, Ash, Protein, Fiber and Fat mean value of different

combination of wheat – mushroom powder biscuit

Treatments Moisture(%) Ash (%) Protein Fiber (%) Fat (%)

(%)

T1 2.39 ± 0.01a 0.84 ± 0.02e 6.61 ± 0.01e 1.45 ± 0.02e 27.61± 0.01e

T2 2.35 ± 0.01b 1.06± 0.01d 6.94 ± 0.01d 1.72 ± 0.01d 29.02± 0.01d

T3 2.31 ± 0.01c 1.32 ± 0.01c 7.40 ± 0.01c 1.95 ± 0.01c 32.12 ± 0.01c

T4 2.23 ± 0.02d 1.59 ± 0.02b 7.82 ± 0.01b 2.17 ± 0.01b 32.44± 0.01b

T5 2.17 ± 0.01e 1.82 ± 0.01a 8.01 ± 0.01a 2.42 ± 0.01a 33.56 ± 0.02a

Pr > F <.0001

The values are means of five replicates ± Standard Error

Mean values with the same superscript letters within the same column do not differ
significantly 5% level

4.4.1 Moisture content

The moisture content of biscuits gradually decreased from 2.39 to 2.17 %. there was a

significant difference in moisture content among the five treatments. According to

DMRT, 100% wheat flour added biscuit (T4) showed a significantly highest mean

value (2.39 ± 0.01) and 20% mushroom powder added biscuit (T5) has the least mean

value (2.17 ± 0.01) of moisture content.

60
 3

2.5
Moisture content %
2

1.5

0.5

0
T1 T2 T3 T4 T5

Figure 4.2: Moisture content of each treatment

4.4.2 Ash content

2.5

2
Ash content %

1.5

0.5

0
T1 T2 T3 T4 T5
Figure 4.3: Ash content of each treatment

The ash content of biscuits gradually increased from 0.84 to 1.82% there was a

significant difference in ash content among the five treatments. According to DMRT,

20% of mushroom powder added biscuit (T5) showed a significantly highest mean

value (1.82 ± 0.01) this may be because the mushroom powder contains a high amount

61
of minerals than wheat flour so the ash content also increased and 100% wheat flour

biscuit (T1) has the least mean value (0.84 ± 0.02) of ash content.

4.4.3 Protein content

9
8
7
Protein content %

6
5
4
3
2
1
0
T1 T2 T3 T4 T5
Figure 4.4: Protein content of each treatment

The protein content of biscuits gradually increased from 6.61 to 8.01% there was a

significant difference in protein content among the five treatments. According to

DMRT, 20% of mushroom powder added biscuit (T5) showed a significantly highest

mean value (8.01 ± 0.01) and 100% wheat flour biscuit (T1) has the least mean value

(0.84 ± 0.02) of protein content. Protein content of mushrooms depends on the

composition of the substratum, size of pileus, harvest time and species of mushrooms

(Bano and Rajarathnam, 1982). On a dry weight basis, mushrooms normally contain

19 to 35% proteins as compared to 7.3% in rice, 12.7% in wheat, 38.1% in soybean

and 9.4% in corn (Crisan and Sands,1978; Li and Chang, 1982; Bano and

Rajarathnam,1982).

62
4.4.4 Fiber content

2.5
Fiber content %

1.5

0.5

0
T1 T2 T3 T4 T5

Figure 4.5: Fiber content of each treatment

The fiber content of biscuits gradually increased from 1.45 to 2.42% there was a

significant difference in fiber content among the five treatments. According to DMRT,

20% of mushroom powder added biscuit (T5) showed a significantly highest mean

value (2.42 ± 0.01) and 100% wheat flour biscuit (T1) has the least mean value (1.45

± 0.02) of fiber content.

4.4.5 Fat content

The fat content of biscuits gradually increased from 27.61 to 33.56% there was a

significant difference in fat content among the five treatments. According to DMRT,

20% of mushroom powder added biscuit (T5) showed a significantly highest mean

value (33.56 ± 0.02) this may be because the original mushroom powder used in

biscuits formulation contains and 100% wheat flour biscuit (T1) has the least mean

value (27.61 ± 0.01) of fat content. In mushrooms, the fat content is very low as

compared to carbohydrates and proteins. The fats present in mushroom fruiting bodies

63
are dominated by unsaturated fatty acids (Singer,1961). Hugaes (1962) observed that

mushrooms are rich in linolenic acid which is an essential fatty acid.

40

35

30

25
Fat content %

20

15

10

0
T1 T2 T3 T4 T5

Figure 4.6: Fat content of each treatment

4.5 CHANGES IN QUALITY CHARACTERISTICS OF WHEAT FLOUR –

MUSHROOM POWDER COMPOSITE BISCUITS DURING STORAGE

4.5.1 Nutritional qualities of mushroom biscuits during storage

Based on the nutritional and sensory analysis of freshly made mushroom biscuits, the

most preferred biscuits were selected for storage studies. These biscuits were packed

in sealed aluminum foil which is commercially used to pack the biscuits. Biscuit packs

were stored under ambient conditions of the average temperature of 30℃ and relative

humidity 75 – 80%

Treatment code Treatments

234 (T1) Biscuits made by 100% Wheat flour

326 (T3) 10g mushroom powder / 100g mixture (10+90g)

425 (T4) 15g mushroom powder / 100g mixture (15+85g)

64
4.5.2 Moisture content of mushroom biscuits during Storage period

Biscuits are very hygroscopic. Therefore, they must be protected from the atmosphere

to prevent or delay the moisture pick up. The moisture content of food products is

changed when there is a moisture gradient outside and inside of the product. Fennema

(1996) stated that low and intermediate moisture foods, such as bakery products, the

ability of proteins to bind the water is critical to the acceptability of these foods.

8
7
Moisture content %

6
5
4 T1
3 T3
2 T4
1
0
2 4 6 8
Storage period (weeks)

Figure 4.7: changes in Moisture content during the storage period

According to the DMRT, moisture content was increased significantly (p<0.05)

throughout the storage period was shown in figure 4.7 There was a significant

difference in the moisture content of T1, T3 and T4 treatments in 2nd, 4th, 6th and 8th

weeks of the storage period.

4.5.3 Ash content of mushroom biscuits during the storage period

The Ash content of the product is related to the mineral composition of that. The

changes in the ash content of the biscuits during storage were shown in the figure. The

ash content of all the treatments was slightly decreased during the storage period.

65
According to DMRT, there were no significant differences in each treatment

throughout the storage period.

4
Ash content %

2
T4
T3
1
T1
0
2 4 6 8
Storage period (Weeks)

Figure 4.8: Changes in Ash content during the storage period

Ash content was decreased significantly (p<0.05) throughout the storage period was

shown in figure 4.8 There was a significant difference in ash content of T1, T3 and T4

treatments in 2nd, 4th, 6th and 8th weeks of the storage period.

4.5.4 Protein content of mushroom biscuits during Storage period

Changes that occur in proteins during processing and storage are endogenous and

exogenous enzymatic activities, chemical reaction and modifications, pH changes, salt

effects, storage fungi contamination, and temperature. Proteins undergo denaturation

by reaction with hydroperoxides and produce objectionable odors and off-flavor

developed during storage. Finot (1990) considered that the loss of amino acids during

storage is largely the result of the Millard reaction leading to the formation of

complexes resistant to enzymatic digestion. Figure 4.09 showed the change in the

protein content of the biscuits during storage.

66
 25

20
Protein Content %
15
T4
10 T3
T1
5

0
2 4 6 8
Storage Period (weeks)

Figure 4.9: Changes in Moisture content during the storage period

there was a significantly different (p<0.05) in protein content of T1, T4 and T5

treatments during the whole storage period. The protein content of each treatment was

decreased gradually.

4.5.5 Fibre content of mushroom biscuits during Storage period

Processes involving heat – treatment may affect the dietary fiber in different ways.

Increased temperature leads to breakage if weak bonds between the polysaccharide

chain. Reactions during processing that may affect the dietary fiber content and its

properties are leakage into the processing water, the formation of Maillard reaction

products thus adding to the lignin content and formation of resistant starch fractions.

The changes in the total fiber content of the biscuits during storage are shown figure

4.10

67
 2.5

2
Fiber Content %
1.5
T1
1 T3
T4
0.5

0
2 4 6 8
Duration (Weeks)

Figure 4.10: Changes in Fiber Content during Storage Period

According to DMRT, the Fiber content of storage of biscuits were decreased

significantly (p<0.05) throughout the storage period.

4.5.6 Fat content of mushroom biscuits during Storage period

According to DMRT, the fat content of storage of biscuits were decreased significantly

(p<0.05) throughout the storage period. Shortening of fat or oil contributes to the

tenderization of baked products through inhibition of gluten development and starch

gelatinization. This is through a waterproofing effect, possibly due to the complex with

the carbohydrate and/ or protein. Lipid oxidation is one of the major causes of food

spoilage. It is of great economic concern to the food industry because it leads to

development, in edible oil and fat-containing foods, of various off-flavors and off-

odors generally called rancid (oxidative rancidity), which render these foods less

acceptable. The changes in fat of the biscuits during the storage are shown in figure

4.11

68
 34

32
Fat Content % 30

28
T1
26 T3
24 T4

22

20
2 4 6 8
Storage Period (weeks)

Figure 4.11: Changes in Fat Content during Storage Period

4.6 Organoleptic Analysis of Mushroom Biscuits during the storage period

Organoleptic qualities analysis for the wheat flour supplement with mushroom powder

was done after 2 months of storage period by the panel of 30 semi-trained judges. The

panel evaluated colour, texture, taste, flavor and overall acceptability of the biscuits

which were selected by previous sensory evaluation as preferred every treatment. A

seven-point hedonic scale was used for this purpose. Sensory characteristics of biscuits

stored at ambient temperature were changed slightly than freshly made mushroom

biscuits. Because Millard reaction, lipid oxidation, moisture uptake and other chemical

reaction may change the sensory qualities of the biscuits during the storage period.

Berger; Fisek (1970) reported that moisture uptake and gas exchange were the cause

of off-odor development in biscuits. The result was shown in table 4.4

69
Table 4.4: Organoleptic Characteristics of Biscuits after 8 Weeks of Storage at

Ambient Temperature

Treatment Color Texture Flavor Taste Overall

acceptability

T1 3.90 ±0.23b 3.95 ± 0.19b 3.84±0.19bc 4.01 ± 0.33b 4.23 ± 0.17bc

T3 3.85± 0.14b 3.72 ± 0.19b 4.21± 0.16b 4.3 ± 0.14b 4.73 ± 0.16b

T4 4.79± 0.17a 5.05 ± 0.18a 5.03 ±0.17a 5.75 ± 0.13a 5.68 ± 0.11a

Pr > F < .0001

The values are the means of triplicates ± Standard Error.
The means with the small letters are not significantly different from each other at a 5% level
based on Tukey's test. Sensory parameters were measured using a seven-point hedonic scale

According to the above Table, 4.4 biscuit which was made by85% wheat flour

incorporated with 15% of mushroom powder (T4) showed the highest organoleptic

qualities than the other two treatments. All the organoleptic qualities of treatments

were decreased after the storage period because several chemical reactions occurred

during the storage period. Millard's reaction has an impact on sensory qualities

(Fennema, 1996).

4.7 Microbial Characteristics Evaluation

the result of the microbial study for every 2weeks in the storage period, in term of total

plate count of microorganism in the plate it is given in table 4.5

70
Table 4.5 Results of Microbial Study

Treatments Microorganism Storage Period (Weeks)

2nd week 4th week 6th week 8th week

T1 Bacteria - - - -

Fungus - - - -

T3 Bacteria - - - -

Fungus - - - +

T4 Bacteria - - - -

Fungus - - - +

"+" Denotes growth of microorganism and "–" denotes no growth of microorganism

There were no microbial colonies found during the storage periods (2nd, 4th,6th weeks).

However, at the end of the storage period (8th week), a small number of colonies were

observed in biscuits. Process Such as roasting and baking in high temperature

destroyed a large number of microorganisms. Limits of microbial counts have been

recommended in most foods to keep them safe for consumption (Ogunjobi and

Ogunwolu, 2010)

71
 CHAPTER 05

CONCLUSIONS

Many researchers have tried to develop new nutritional products by incorporating new

bioactive compounds and enrichment of proteins into biscuits. Oyster mushrooms have

become attractive as nonconventional food around the globe because of their culinary,

nutritional and medicinal attributes. They are endowed with high nutrition content

which contains a high value of protein, carbohydrates, fiber, minerals, vitamins (B1,

B2, B3, C and D2) and low fat. The study was carried out to study the development

and quality evaluation of protein-enriched biscuits using oyster mushroom powder.

Drying is a good way to reduce the spoilage of mushrooms. Moreover, the surplus

often cannot be stored for sale in the offseason.

Expansion of storage facilities and processing of mushroom into various value-added

products that could be preserved for a long time is the innovative remedy for this

problem. Successful combination oyster mushroom powder with wheat flour for

biscuit production would be nutritionally advantageous and also mushroom powder

with wheat flour has a significant effect on the functional properties of the blends.

Therefore, 100% wheat flour, 10% mushroom powder incorporated biscuits, 15%

mushroom powder incorporated biscuits were selected as the preferred combination

based on the nutritional and organoleptic characters.

The finding of the storage study revealed that the 15% oyster mushroom powder

incorporated biscuits have the highly acceptable functional, nutritional, organoleptic

quality and best shelf life compare to other combinations from the overall acceptance

rating, the 15% oyster mushroom powder has the highest mean value and no

remarkable changes in organoleptic characters were observed up to 2 months of

72
storage in the ambient condition of average temperature 30 and relative humidity of

75 %- 80%, indicating that the 15% mushroom powder added biscuits could be stored

up to 2 months of storage. Biscuits made with 10% mushroom powder also have

certain distinct functional properties that are more or less similar functional

characteristics of baked products made up of wheat flour. Blending mushroom powder

with wheat flour up to 15% level produced biscuits that can be used for the production

of bakery foods with improved functional properties using innovative processing

methods according to their specific and distinct functional and nutritional properties.

When the percentage of supplemented mushroom powder increases, the overall

acceptance rate goes down. It may be due to the mushroom powder that does not

contain gluten protein which plays a major role in leavened bakery products the

viscous-elastic that can retain gas during baking and also when increases mushroom

powder the browning effect has happened so the color also effected during baking.

A mixture of wheat flour and oyster mushroom powder could make a protein-enrich

product and also good baking product, which should increase its economic value.

Improved quality and productivity of oyster mushrooms could substantially reduce the

protein malnutrition mainly for children in developing countries. The outcome of the

present research can be used as valuable information for the development of high

protein biscuits. The result obtained could be very valuable in decision-making for

industries that want to take nutritional advantages of mushroom powder as alternative.

Oyster mushroom powder could be useful in the manufacture of highly nutritious

biscuits.

73
 6.0 SUGGESTIONS FOR FUTURE RESEARCH WORK

 Industrialization studies should be carried out to determine the highest level of

substitution of oyster mushroom powder for wheat flour in the bakery products.

 Development studies needed to prepare different value-added products using

oyster mushroom powder.

 Studies should be carried out to analyze the consumer acceptability of

mushroom-wheat biscuits by the market survey.

 Evaluate the technical feasibility to increase production potential.

74
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