Development of mushroom based sausage.

By
Jayathilake. W.D.

Thesis submitted to the University of Sri Jayewardenepura in the partial fulfillment

requirement for the award of the degree of Master of Food Science and Technology.

MSc. 2008.
 Declaration.

The worked describe in this thesis, development of mushroom based

sausage, its microbiological testes, sensory analysis was carried out in Food
Research Units' Laboratory by me under the supervision of Dr K. H.
Sarananda Head, Food Research Unit, Department of Agriculture
Gannoruwa and its proximate analysis and self life evaluation was carried
out in laboratory of Food Science Department by me under the supervision
of Dr K.K.D.S.Ranaweera Head, Department of food science and
Technology, Coordinator, Food science and Technology post graduate,
programs University of Sri Jayewardenepura Gangodawila Nugegoda and
the report on this has not been submitted in whole or in part to any other
institution for another degree.

Jaya ilake W.D.

 We hereby certify that the above statement made by the candidate is
true and this thesis suitable for submission to the University for the
purpose of evaluation.

Dr. K.H. Sarananda.

Head,
;: K.K.f.S. Ranaweera.
Head,
Food Research Unit, Department of Food Science and
Department of Agriculture, Technology,
Gannoruwa. Coordinator,
Food Science and Technology
post graduate Programs,
University of Sri Jayawardenapura
Gangodawila,
Nugegoda.
 CONTENT
Title pages
Content I
Acknowledgement IV
Abstract V
Tables & Equations IX

Chapter 01. Introduction. 1

Chapter 02. Literature review. 2
2.1. Mushroom. 2
2.1.2. Edible mushrooms. 2
2.2. Cultivation. 4
2.2.1. How are mushrooms grown. 4
2.3. Usage 5
2.3.1. Value and nutrition. 6
2.4. Ingredients for sausages production. 6
2.4.1. Binder. 6
2.4.2. Filler. 6
2.4.3. Salt. 6
2.4.4. Sugar. 7
2.5. Microbiology. 7
2.5.1.Coliform. 7
2.6. Casing. 7
2.7. Proximate analysis. 8
2.7.1 Moisture. 8
2.7.2. Fat. 8
2.7.3. Fibre. 8
2.7.4. Protein. 8
2.7.5. Ash. 8
Chapter 03.
3.1. Material and methodology. 9
3.2. Dehydration and dehydrated powder of mushroom for
sausages 9
 3.2.1. Material. 9
3.2.2. Methodology. 9
3.3. Fresh mushroom for sausages. 9
3.3.1. Materials. 9
3.3.1.2. Raw material quality. 10
3.3.1.3. Usage of fresh mushroom 10
3.3.2. Other ingredients. 10
3.3.3. Methodology. 10
3.3.4. Mushroom base sausage development. 12
3.3.5. Final product. 15
3.3.5.1. Microbiology. 15
3.3.5.2. Coliform. 15
3.3.5.3. Total plate count (TPC) 16
3.4. Keeping quality determination. 16
3.5. Sensory evaluation. 17
3.5.1. Materials. 17
3.6. Proximate analysis. 17
3.6.1. Moisture. 17
3.6.1.1. Oven drying method. 17
3.6.2. Determination of Total Ash. 18
3.6.3. Fat. 18
3.6.3.1. Determination of free fat by Soxhlet
Extraction Method. 18
3.6.3.2. Determination of Total Fat 19
3.6.4. Crude fibre. 20
3.6.5. Protein. 21
3.7 Cost analysis. 22
Chapter 04. 23
4.1. Results and discussion. 23
4.1.1. Mushroom powder. 23
4.1.2. Dehydrated flesh. 23

11
 4.1.3. The final product. 23
4.1.3.lQuality of the final product. 23
4.2 Collection of mushroom. 23
4.3 Processing of other ingredients. 24
4.4 Method used for processing mushroom sausages 24
4.5 Spices. 24
4.6. Processing steps. 25
4.7. Result of microbiology. 26
4.7.1. Coliform. 26
4.7.2. Total plat count. 26
4.8. Result and calculation of proximate analysis. 26

Chapter 05 27
5.1. Conclusion. 27
5.2 Recommendation and suggestions. 27
Reference VI

111
 Acknowledgement.
First and foremost I wish to express my deepest sense of gratitude
to both my internal supervisor Dr.K.K.D.S.Ranaweera Head,
Department of Food Science and Technology, Coordinator, Food
Science and Technology post graduate programs University of Sri
Jayewardenepura for his valuable advices, encouragement and
guidance throughout this study and for reading the manuscript and
sparing his valuable time in bringing this study to a successful
completion and my external supervisor Dr. K.I-I.Sarananda Head,
Food Research Unit, Department of Agriculture, Gannoruwa,
Peradeniya, guidance given in carried out this research project.
I am also grateful to Mr. Jagath Wansapala, Mrs. Indira
Wickramasinghe Mrs. Rupika perera and all staff members of
lectures and laboratory staff of the university and Mr. Senarath
Ekanayake, Mr. Bandula Jayasinghe, Mr. Janaka wijesinghe and all
other staff and miner staff of the Food Research Unit.
Finally my greatest thanks are being to all the personnel who
encourage me during this research work.

lv
Development of Mushroom based sausage by
Jayathilake W.D.

Abstract

Oyster mushroom (Pleurotus ostreatus) is a common mushroom cultivated in Sri Lanka

and is prized for its edibility and texture. Oyster mushroom has a high nutritional value
due to its high level of vitamins and proteins and its non-saturated fafty acids. The
consumption of Oyster mushroom reduces cholesterol levels remarkably and has also
been attributed anticancer properties. Diversification of value added products of
mushrooms can be one of the successful approaches to popularize mushroom
consumption among Sri Lankans.

A study was carried out to develop a mushroom based vegetable sausage formula and to
determine its self life/keeping quality, microbial quality and proximate analysis.

Oyster mushroom was dehydrated and made powder, dehydrated flesh rehydrated with
hot water and then made pulp and pulp of fresh Oyster mushroom were used to prepare
the sausage formula. According to the analysis of microbiological and sensory quality,
the final product kept in cold room storage for more than four months, it has been
revealed that the ingredients used were complementarily suitable to each other and the
conditions used to store were appropriate for the final product. Self life of the final
product is longer for more than two months and seems to be much longer. According to
quality characteristics of final product, it can be assumed that the mushroom formula
satisfies the sensory assessments.

New processing operations like use of pre cooked mushroom flesh with spices mixture
are recommended to introduce to enhance the flavor profile of the final product.
Chapter 01
1. Introduction.
Production and consumption of sausages have been started over many centuries ago and
still their major raw ingredients are simply meat fish or both. Meat or fish based sausages
can be therefore consumed only by consumers who consume meat or fish but cannot be a
commodity suitable for vegetarians. On the other hand, people tend to become vegetarians
due to many reasons.

Hence, there is potential for developing vegetable based sausages which can be of high
demand in both non-vegetarians and vegetarians. In order to achieve this, it is necessary to
identify ingredients suitable for the sausage formula, which are also locally available. The
other major requirements are that the product developed should be nutritious and palatable.
The cultivation of raw materials should be easy and cheaper.

Mushrooms can be used as a suitable raw material which meets the above requirements and
requires a smaller space for its cultivation. On the other hand, this value addition method
will minimize the postharvest losses of the commodity. Especially, Oyster mushroom
(Pleurotus ostreatus) is a common mushroom cultivated in Sri Lanka and is prized for its
edibility and texture. Oyster mushroom has a high nutritional value due to its high level of
vitamins and proteins and its non-saturated fatty acids. The consumption of Oyster
mushroom is said to reduce cholesterol levels and has also been attributed anticancer
properties. Very-low-density lipoproteins and low-density lipoproteins are reported to be
reduced in the total reduction of serum cholesterol. Cholesterol content in high-density
lipoproteins was not significantly affected by oyster mushroom.

Therefore, the main objectives of the study were to develop a mushroom based vegetable
sausage formula and to determine its self life/keeping quality, microbial quality and
proximate analysis.

1
Chapter 02.
2.0 Literature review.
2.1. Mushroom.
A mushroom is the fleshy, spore-bearing fruiting body of a fungus, typically produced
above ground on soil or on its food source. The standard for the name "mushroom" is the
cultivated white button mushroom, Agaricus bisporus, hence the word mushroom is most
often applied to fungi (Basidiomycota, Agaricomycetes) that have a stem (stipe), a cap
(pileus), and gills (lamellae, sing. lamella) on the underside of the cap just as do store-
bought white mushrooms. However, "mushroom" can also refer to a wide variety of gilled
fungi, with or without stems, and the term is used even more generally to describe both the
fleshy fruiting bodies of some Ascomycota and the woody or leathery fruiting bodies of
some Basidiomycota, depending upon the context of the word. Forms deviating from the
standard form usually have more specific names, such as "pufThall", "stinkhorn", and
"morel", and gilled mushrooms themselves are often called "agarics" in reference to their
similarity to Agaricus or their placement in the order Agaricales. By extension,
"mushroom" can also designate the entire fungus when in culture or the thallus (called a
mycelium) of species forming the fruiting bodies called mushrooms. (Alice Beetz and
Michael Kustudia 2003)

2.1.2. Edible mushrooms.

Aaricus bisporus, also known as champignon and the button mushroom. This species also
includes the portobello and criniini mushrooms.

Agaricus campestris - Meadow mushroom.

A uricularia polytricha or A uricularia auricula-judae (Tree ear fungus), two closely related
species of jelly fungi that are commonly used in Chinese cuisine.

Flammulina velutipes, the "winter mushroom", also known as enokitake in Japan.

2
Ilypsizvzus tessulatus (also Hypsizvjus marinoreus), called shimeji in Japanese, it is a
common variety of mushroom available in most markets in Japan. Known as "Beech
mushroom" in Europe.

,, also known as shiitake, oak mushroom. Lentinus edodes is largely produced in Japan,
China and South Korea. Lentinus edodes accounts for 10% of world production of
cultivated mushrooms. Common in Japan, China, Australia and North America.

Pleurotus species - Pleurotus is a genus with a number of distinct species and strains.
Pleurotus species are characterized by a white spore print, attached to decurrent gills, often
with an eccentric (off center) stipe or no stipe at all. The oyster mushroom and king
trumpet mushroom and is popular and delicious. Pleurotus mushrooms are the second most
important mushrooms in production in the world, 25% of total world production of
cultivated mushrooms. The common name "oyster mushroom" comes from the white shell-
like appearance of the fruiting body, not from the taste. The taste of the oyster mushroom
varies from very mild to very strong. It varies in texture from very soft to very chewy,
depending on the strain. Pleurotus mushrooms are world-wide, China is the major
producer. Several species can be grown on carbonaceous matter such as straw or
newspaper. In the wild they are usually found growing on wood. It contains Vc, and
microelements such as P, K, Te, Zn, Cu,, Co. Mo, and abundant amino acids --especially
glutamic acid. Besides being eaten, it can be used to ease the body, decrease cholesterin,
lower blood pressure, guard against arteriosclerosis and against tumors.

Pleurotus ostreatus (common oyster mushroom).

Pleurotus ostreatus (common oyster mushroom) forms shelves or clusters of fairly long-
stemmed fungi whose caps are cream to taupe and rounded or spoon-shaped. This
mushroom ranges in size from a gumdrop to a saucer, covering sharp gills. All varieties are
relatively tender and mild when cooked. These are by far the easiest and least expensive to
grow; however, many growers are now using straw as the growth medium. This results in a
loss of flavour, texture, and shelf-life. The natural host is wood of varying, yet similar,
types, which produce a more defined body and character.

3
Different substrates can be used to cultivate Pleurotus ostreatus and to get a successful
yield (Baysal, et al 2003; Reddy, R 2000).

Rhizopus oligosporus - the fungal starter culture used in the production of tempeh. In
tempeh the mycelia of R. oligosporus are consumed. Sparassis crispa - recent
developments have led to this being cultivated in California. Tremella fucformis (Snow
fungus), another type ofjelly fungus that is commonly used in Chinese cuisine.

Tuber species, (the truffle), Truffles belong to the ascomycete grouping of fungi. The
truffle fruitbodies develop underground in mycorrhizal association with certain trees e.g.
oak, poplar, beech, and hazel. Being difficult to find, trained pigs or dogs are often used to
sniff them out for easy harvesting.

Ustilago maydis (Corn smut), a fungal pathogen of the maize plants. Also called the
Mexican truffle, although not a true truffle.

Volvariella volvacea (the "Paddy straw mushroom.") Volvariella mushrooms account for
16% of total production of cultivated mushrooms in the world.

2.2. Cultivation.
2.2.1. How are mushrooms grown.

There are many ways to grow mushrooms, but productions always occurs in three general
steps—spawn run, pinning, and fruiting. Spawn run is the complete colonization of a
suitable substrate following inoculation of the substrate, called spawning. The second step,
pinning, is the stage of growth when pitheads are initiated. Pinheads are knots of mycelium
that eventually develop into mushrooms. All species of mushrooms require a set of
environmental conditions for pinning that are different from the conditions for optimum
mycelial growth. Most, if not all, cultivated mushrooms fruit at lower temperatures than the
optimum for substrate colonization. The last step, fruiting, is the development of the pins
into mature mushrooms.

In
Step I. Spawn Run: After the substrate is prepared and sterilized in the plastic bag or other
suitable container, spawn is aseptically added. Spawn is typically grown on sterilized grain
(usually rye or millet) and is either produced by the cultivator or purchased from a
mushroom supply house. Purity of the spawn is absolutely critical. In most specialty
mushrooms, spawn is added at a rate of 2.5% or more of the dry weight of the substrate.
After the bag is hermetically sealed, the spawn is evenly distributed throughout the sawdust
by shaking the bag. Depending on the species of mushroom, the substrate is usually fully
captured by the mycelium within 2 to 6 weeks. Spawn run temperatures should be 2 1-24°C.

Step II. Pinning: To initiate mushroom formation, temperatures are dropped for 2 days to 2
weeks, CO2, levels are lowered by introducing fresh air, and light is provided (if you can
read by it, there is enough light). Often, the bags are simply opened and moved to the
growing room.

Step III. Fruiting: The colonized bag of substrate is placed in a growing room maintained at
cool temperatures (63°F is a good average for most mushrooms) and high relative humidity
(85-95%). Although the common button mushroom does not require light, all of the other
mushrooms described here need some light for proper development. For most specialty
mushrooms, the bag may be removed from the sawdust block after spawn run is complete.
It is often desirable, however, to remove only the top half of the bag so the sides of the bag
reduce air movement across the top of the block, thus maintaining high humidity. In the
production of Oyster and Lion's Mane mushrooms, holes are cut in the plastic bag and the
mushrooms are allowed to grow through the holes. In Shiitake production, the plastic bag is
usually completely removed since this mushroom develops a tough skin on the surface of
the sawdust substrate.

2.3. Usage
Fresh.
Dehydration and dehydrated powder of mushroom.
Product.
Medicinal and pharmaceutical.

61
2.3.1. Value and nutrition.
Main articles: Edible mushrooms, Mushroom hunting, and Fungi culture.

Edible mushrooms are used extensively in cooking, in many cuisines (notably Chinese,
European, and Japanese). Though mushrooms are commonly thought to have little
nutritional value, many species are high in fiber and provide vitamins such as thiamine,
riboflavin, niacin, biotin, cobalamins, ascorbic acid. Though not normally a significant
source of vitamin D, some mushrooms can become significant sources after exposure to
ultraviolet light, though this also darkens their skin. Mushrooms are also a source of some
minerals, including iron, selenium, potassium and phosphorous.

2.4. Ingredients for sausages production.

Here major ingredients are fresh mushrooms and other ingredients are commonly known as
binders, fillers, and extenders. Many countries have restrictions on type, amount, and
quality of ingredients used in sausages production.

2.4.1. Binder.
Binders are protinaceous agent which changing water binding properties which help
binding together different material in sausage product. Sometime they also contribute to the
fat emulsification.

2.4.2. Filler.
Carbohydrate products which are used to absorb excessive water and not much affect to
emulsification properties. Common fillers are cereal flour, starches derived from corn, rice,
potato etc.

2.4.3. Salt.
Salt is an essential ingredient of any sausage formulation. Salt is used to preserve the
product, enhance the flavor, and or solubilize the meat proteins in order to improve the
binding properties of the formulation. Since the advent of refrigeration, the preservative
properties are the least important use of salt, though dry sausages still use salt for
preservation. The most important use of salt in a sausage product is its ability to solubilize

6
proteins. This enhances the product texture and improves water and fat binding. Since
sodium chloride (NaCl) salt has been linked to hypertension, other non-sodium salts, such
as potassium and calcium chlorides, are sometimes substituted for a portion of the sodium
chloride.

2.4.4. Sugar.
Sugars are used in sausage formulations to reduce the flavor intensity of the salt and
flavorings, and to provide a food source to enable microbial fermentation. Sugars used in
sausage products include sucrose and dextrose.

2.5. Microbiology.
2.5.1. .Coliform.
Coliform is a leading cause of food borne illness. Coliform organisms can be eliminated
from cooked sausages by proper cooking processes.

2.6. Casing.
Regenerated collagen sausage casings are made from collagen extracted from cattle hides
and hog skins in a process called regeneration. The extracted collagen is dissolved, and
then hardened, washing, swelled with acid, and finally formed into the tubular casing shape
in an extrusion process. This final shape is then fixed in an alkali bath. These types of
lower strength casings are typically used for smaller diameter products. Synthetic or
artificial casings are made from special papers impregnated with cellulose, saran casings
made from synthetic plastics, and hydro-cellulose casings made from regenerated cellulose.
Cellulose casings are created from dissolved fibers extracted from cotton seeds or paper
pulp. Each of these types of casings is available in a wide range of sizes and characteristics
and is easy to handle, however, these types of casings are not edible and must be removed
from the sausage prior to consumption. Artfficial casings provide high strength and are
available with excellent permeability to moisture and smoke, or as impermeable casings for
use in producing water-cooked products such as braunschweiger.

7
2.7. Proximate analysis.
2.7.1 Moisture.
The moisture content of foods varies widely. The water molecules in a food may be present
in a variety of different molecular environments depending on their interaction with the
surrounding molecules. The water molecules in these different environments normally
have different physiochemical properties:

2.7.2. Fat.
Lipids are one of the major constituents of foods, and are important in our diet for a number
of reasons. They are a major source of energy and provide essential lipid nutrients.
Nevertheless, over-consumption of certain lipid components can be detrimental to our
health, e.g. cholesterol and saturated fats. In many foods the lipid component plays a major
role in determining the overall physical characteristics, such as flavor, texture, mouth feel
and appearance.
2.7.3. Fibre.
The crude fiber consists largely of cellulose together with a little lignin or unavailable
carbohydrates

2.7.4. Protein.
Proteins are an abundant component in all cells, and almost all except storage proteins are
important for biological functions and cell structure. Food proteins are very complex

2.7.5. Ash.
Ash refers to the inorganic residue remaining after either ignition or complete oxidation of
organic matter in food stuff. Ash content represents the total mineral content in foods.
Determining the ash content may be important for several reasons. It is the part of the
proximate analysis for nutritional evaluation. Ashing is the first step in the preparation of a
food sample for specific elementary analysis. Because certain foods are high in particular
minerals, ash content becomes important.
Chapter 03.
3.1. Material and methodology.
3.2. Dehydration and dehydrated powder of mushroom for sausages
3.2.1. Material.
Mushroom.
Citric acids.
Potable water.
Knife.
Electric dryer.
Loading trays.
Cleaning baskets.

3.2.2. Methodology.
Dry cleaned good quality fresh American oyster verity was selected from the farm gate.
Available at the farm gate trimmed dry cleaned well packed in a suitable container for
buying appropriate quantities with 200g up to 5 kg. 3 kg of oyster fresh was collected and
brought to the food research unit laboratory. Observed, cleaned and while making strips
about 1cm to 11/2cm of width and dip in a 3% citric acid solution where previously
prepared and drain the solution and uniformly loaded on trays. The trays were kept in an
electric oven at which temperature about 60°C ±2.
After about 7 hours in an oven take them out and make powder and weighed out the
quantity of yield. Some quantity of dehydrated flesh without making powder dehydrated
and then used as raw major ingredient for making sausages.

3.3. Fresh mushroom for sausages.

3.3.1. Materials.
Selected American oyster species was used other species also can used but cheap and
wastages is minimum with oyster species.

9
3.3.1.2. Raw material quality.
Selected one brand only used for preparing sausage throughout this experiment

3.3.1.3. Usage of fresh mushroom.

Make paste without adding water or iced or other ingredients. The paste was used without
drain to prepare sausages

33.2. Other ingredients.

Binders and fillers.
According to literature can use many protinaceous agents which animal or pant origin but
here both binding as well as filling agent was corn starch which relatively cheap and
readily available for vast production.

Spices
Pepper, chilly, turmeric and clove were used in powder foam and others cinnamon,
cardamom, nutmeg, onion, garlic, were cleaned and ground to very fme past without
remaining of fibre parts in paste.
Salt
Sugar
Sodium tri phosphate
Casings
Knife
Steamer
Filling nozzle
Cold storage facilities and suitable thread.

3.3.3. Methodology.
Powder formed of spices, other ingredients were weighted out according to the formula
which was going to prepare. Other spices also weighted and chopped and fmely ground
using mortar and pestle. Fresh oyster mushroom ground to make a paste and soon it
transferred and mixed with salt and sodium tri phosphate. Ground spices were mixed

10
together. Then other powder formed spices were added to the mixture while mixing the
stuff and then the mixture of sugar and corn starch were added the mixture and then
homogeneously mixed the stuff to make mushroom base sausages.
Thereafter mixture was inserted to the filling nozzle and then filled in to food grade
suitable diameter casing which was one end previously tied by itself. The air which
enwrapped during processing and filling was removed. The remaining end was tied to as
much as possibly to compress the mixture. Then middle of the casing was tied by using
thread to make it more compressed. Again and again middle of casing should tie to get
appropriate length of one pies of sausage. It's easy for filling, removing wrapped air and
compressed more when the length of casing enough for two or four pieces of sausages.

The sausages were kept in a previously heated steamer and steamed them about 17 to 20
minutes with twist opening the lid of the steamer during steaming. Sausages were taken out
from the steamer and cooled them by running water. Then air dried sausages were stored in
cold room where temperature adjusted previously bellow 0°C.

11
3.3.4. Mushroom base sausage development.
According to the method described above many of formula were prepared to obtain the
fmal product formula.
Table #1 Development of mushroom based sausages formula
Ingredients Grams
Mushroom 300
Corn starch 15
Salt 6
Sodium try phosphate 1.5
Chilly powder 3
Pepper powder 3
Turmeric powder 2
Garlic 3
Onion X
Clove X
Cardamom X
Cinnamon X
Nutmeg X

12
Table #2 Development of mushroom based sausages formula
Ingredients Grams Grams Grams
Mushroom 300 300 300
Cornstarch 18 21 25
Salt 7.5 7.5 7.5
Sodium try phosphate 1.5 1.5 1.5
Chilly powder 5 5 5
Pepper powder 4.5 4.5 4.5
Turmeric powder 2 2.5 3
Garlic 3. 3 3
Onion 5 5 5
Clove 2.5 2.5 2.5
Cardamom 0.5 0.5 0.5
Cinnamon 0.5 0.5 0.5
Nutmeg 0.25 0.25 0.25
Table #3 Development of mushroom based sausages formula
Ingredients Grams Grams Grams
Mushroom 300 300 300
Corn starch 22 23 24
Salt 7.5 7.5 7.5
Sodium try phosphate 1.5 1.5 1.5
Chilly powder 5 5 6
Pepper powder 4.5 4.5 4.5
Turmeric powder 3 3 3
Garlic 3 3 3
Onion 5 5 5
Clove 1.5 1.5 1.5
Cardamom 0.5 0.5 0.5
Cinnamon 0.5 0.5 0.5
Nutmeg 0.25 0.25 0.25

13
Table #4 Development of mushroom based sausages formula
Ingredients Grams Grams Grams
Mushroom 300 300 300
Corn starch 24 24 24
Salt 7.5 7.5 8
Sodium try phosphate 1.5 1.5 1.5
Chilly powder 5 5 6
Pepper powder 4.5 5 6
Turmeric powder 3 3 3
Garlic 3 3 3.5
Onion 5 5.5 6
Clove 1.5 1.25 1
Cardamom 0.5 0.25 0.25
Cinnamon 0.5 0.5 0.4
Nutmeg 0.25 0.24 0.24
Table #5 Development of mushroom based sausages formula
Ingredients Grams Grams
Mushroom 300 300
Corn starch 24 24
Salt 8 7.5
Sodium try phosphate 1.5 1.5
Chilly powder 6 5
Pepper powder 6 4.5
Turmeric powder 3 2.75
Garlic 3.5 3.25
Onion 6 5.5
Clove 1 0.8
Cardamom 0.25 .25
Cinnamon 0.4 0.45
Nutmeg 0.24 0.24

14
Table #6 Development of mushroom based sausages formula
Ingredients Grams
Mushroom 300
Corn starch 24
Salt 7.5
Sodium try phosphate 1.5
Chilly powder 5
Pepper powder 4.5
Turmeric powder 3
Garlic 3.25
Onion 5.5
Clove 0.8
Cardamom 0.25
Cinnamon 0.45
Nutmeg 0.24

3.3.5. Final product.

According to sensory test basic formula was evaluated up to the fmal product which self
life and microbiology was conducted weekly with first two month and then once a time for
two weeks.
3.3.5.1. Microbiology.
Total plate cont and Coliform were tested according to Sri Lankan Standard and AOAC
approved method which means of using of 3M Petri film respectively.
Well cleaned glass Petri disk, pipettes, test tube with caps which covered with aluminum
foils, flasks with cotton plugs, instruments for preparation samples, sterile and nutrient agar
solution which prepared accordance with manufacture instruction provided were
autoclaved at 121°C (15 psi) for 15 minutes.
3.3.5.2.Coliform.
As describe above equipment needed for coliform enumeration were sterile except glass
petri disks because here instead of Petri disks and media used 3M Petri films.

15
Thawn sausage sample of 1 Og was weighed and sample was transferred to 10 ml sterile
distilled water (This is called 100 dilution)
This mixture was kept in a sterile stomacher blender and it was blended for 5 minutes.
Sterile distilled water 9m1 was taken into test tube and 1 ml of water sample from dilution
100 and it was shacked gently for 2 minutes by rotating it between the palms. (10 dilution)
Another 9ml-distilled water was taken and added lml from earlier one. (102 dilution)
Similarly dilution series were prepared up to iø - .
Transferred aliquots of 1 ml from each, 100' 10-1, 10-2and10 3 dilution separately on the
3M Petri films. And triplicate.
All the 3M Petri films were incubated upside-down at 37°C for 18 to24 hours. This is
practiced weekly with first two month and then once a time for two weeks.

3.3.53. Total plate count (TPC)

Thawn sausage sample of I Og was weighed and sample was transferred to 10 ml sterile
distilled water (This is called 100 dilution)
This mixture was kept in a sterile stomacher blender and it was blended for 5 minutes.
Sterile distilled water 9m1 was taken into test tube and I ml of water sample from dilution
100 and it was shacked gently for 2 minutes by rotating it between the palms. (10-' dilution)
Another 9 ml- Sterile distilled water was taken and added imi from earlier one. (10-2
dilution). Similarly dilution series were prepared up to 10-3 . Transferred aliquots of I ml
from each, 100' 10-1,1 0-2and I 0 dilution separately into sterile Petri dishes in triplicate and
poured 15 ml of sterilized nutrient agar medium in each plate. All the plates were incubated
upside-down at 370C for 48-72 hours. This is practiced weekly with first two month and
then once a time for two weeks.

3.4. Keeping quality determination.

Final product kept on a plastic tray and wrapped by polythene here unable to practice
vacuum packing system and stored in cold room. Self life of the product was determined by
mainly microorganism which continuously enumerate and observation and evaluation of
appearance, texture, color, smell and flavor of the mushroom sausages.

16
3.5. Sensory evaluation.
3.5.1. Materials.
Panel of sensory test.
Pencils
Score sheets
Trays
Cups of water
Available two brands of meat sausages in local market and mushroom sausages fried in
coconut oil. Sensory characteristics such as colour, appearance, taste and texture were
evaluated when finalized the formula and developing the product as well as during the self
life determination and also final product sensory evaluation at which has fmished four
month of storage time in cold room.

All The panel members from staff of food research unite and the overall acceptability was
evaluated on a seven point grading scale.(7=like very much, 6=like moderately, 5=like
slightly,4=neither like nor dislike, 3=dislike slightly,2= dislike moderately, 1 = dislike very
much) the scorecard was prepared to estimate overall quality acceptance of mush room
sausage by organoleptic method. Prepared card see on appendix 01

3.6. Proximate analysis.

3.6.1. Moisture.
3.6.1.1. Oven drying method.
Materials:
Moisture dish made of stainless steel
Oven maintained at 105°C
Weighing balance
Procedure:
Previously oven dried three moisture dishes were taken.
About 5 g of the mushroom sausage sample was weighed to the each dish.
Uncovered dishes were dried at 105°C for four hours.

17
Then dishes were covered with lid and transferred to the desiccators and weighed when
dishes were cooled.
Heating and weighing was repeated until constant weight obtained.

Ash
3.6.2. Determination of Total Ash.
Materials:
Muffle furnace
Silica dishes
Weighing balance
Desiccators
Tongs
Gloves (thermal)
Procedure:
About 5 g of the sample was weighed in to a clean and dry pre weighed porcelain dish.
Sample was slowly ignited over an incinerator.
The dish was transferred to the muffle furnace set at 550°C and incinerated until it was free
of black carbon particles and until it was white in colour.
The dish was removed carefully and let cool in a desiccator. After cooling, dish was
weighed. Above procedure was done for other two samples.

3.6.3. Fat.
Free fat.
3.6.3.1. Determination of free fat by Soxhlet Extraction Method.
Materials:
Soxhlet extraction apparatus ( Flask extractor, thimble, & condenser)
Heating mantle/heating water baths
Weighing scale
Diying oven
Petroleum ether
Pumic chips

FI:
Procedure:
About 5 g of fmely chopped sample was weighted and added anhydrous sodium sulphate.
Sample was ground until fme powder obtained. All powdered materials in the motor were
transferred in to extraction thimble and the end of the thimble was plugged with cotton
wool.
Extraction thimble with sample was placed in the apparatus.
Cleaned and dried round bottom flask was weighed and 200 ml of petroleum ether was
added to the flask.
The flask was connected to the Soxhiet extractor and was fitted the condenser.
Sample was refluxed for three hours.
When the refluxing was over distilled off the solvent and flask was placed on water bath.
When the solvent was evaporated, flask was kept in the oven for two hours.
Then flask was taken out, cooled and weighed. This process was repeated until constant
weight obtained.
Repeated the procedure for another 2 samples too.

3.6.3.2. Determination of Total Fat.

By Mojonnier extraction Method
Materials:
Beakers (100 ml)
Majonnier flask
Petroleum ether
Hydrochloric acid (prepared by mixing 25m1 of conc.HC1 with 1 ml of water)
Ethanol
Diethyl ether

Procedure:
About 2 g of the sample was placed in a 100 ml beaker lml of NH3 added to the beaker and
contents were mixed thoroughly. Then the beaker was placed on a water bath (70°C - 80
°C) and the content was stirred for about 30 -40 minutes frequently. Then the beaker was

19
removed from the water bath and cooled to at room temperature. About 10 ml of ethyl
alcohol was added to it and the mixture was transferred into the majonnier flask.
The beaker was washed with a 25 ml of diethyl ether in three portions and it was added to
the flask. Then the flask was shaken vigorously for about 1 mm. To the flask 25 ml of pet
ether was added and shake again for one minute. The flask was kept until the upper ether
layer is cleared. The upper ether layer was taken into clan dried weighed flask. Then it was
dried in a water bath at 80 °C until a constant weight was obtained.
Three samples were carried out above procedure simultaneously.

3.6.4. Crude fibre.

Determination of Crude Fiber
Materials:
Round bottom flask
Beaker
Reflux condenser
Sintered crucible (gooch crucible)
Asbestos wool
Desiccator
Heating Mantle
Weighing balance
Dii. Sulphuric acid
Sodium Hydroxide
Litmus papers
Procedure:
About 2 g of the defatted sample was weighed and transferred to a 1 L conical flask.
About 200m] of boiling 1.25 % Sulphuric acid was added and boiled for 30 min over a
Bunsen burner. Flask was occasionally swirled to remove solids from adhering to the sides
of the flask. The hot solution was decanted through a Sintered crucible fitted with vacuum
exhauster. All the residues were transfer and rinsed with boiling water. About 200 ml of
boiling 1.25 % Sodium hydroxide solution was used to transfer the residue from the
Sintered crucible into the flask. And it was boiled for 30 mm.

20
Solution was filtered through previous sintered crucible and washed with 1 % HCl until the
filtrate was free from alkali and then wash with distilled water and small amount of alcohol
and diethyl ether.
The crucible dried in the oven
Then it was cooled and weighed.
The crucible was incinerated at 500 °C, then cooled and weighed.

3.6.5. Protein.
Materials:
Kjeldhal digestion kit ( Kjeldhal digestion flasks(30m1), Kjeldhal heater, Fume trap,
Distillation unit-Paranas Wagner still))
Titration flask
Weighing balance
Sodium sulphate solution
4 % Boric acid solution
0.02 M HCL solution
Kjeldhal catalyst tablets (Mercury catalyst)
Indicators: Methyl red, Methylene blue (prepared a solution by mixing two parts of 2%
methyl red (alcoholic) solution with one part of 0.2% (alcoholic) methylene blue solution)

Procedure:
Four kjeldhal flasks were taken and one was kept as 'blank' sample. A sample of 1 g
prepared sample on a tissue paper was transferred to other three kjeldhal flask. A tablet of
catalyst and 2 ml of sulphuric acid was added to each flask. The flasks were connected to
the fume trap and attached to the pump. The samples were digested for about 1 hour until a
clear solution without black particles was obtained. Then flasks were cooled for one hour.
About 5 ml of 4% boric acid solution and 3 drops of indicator was added into a titration
flask. The sample was dissolved with a minimum amount of ammonia free distilled water
and transferred it to a semi micro kjeldhal distillation apparatus, which had been previously
conditioned by passing steam through it for several minutes slowly. About 8 ml of Sodium
hydroxide & Sodium sulphite solution was added to flask.

21
Then titration flak with boric acid solution was kept at the end of the digestion apparatus to
trap the ammonia liberated. Steam was passed through the flask until about 15 ml of
distillate was received. This solution was titrated with the standard HCL solution until pink
colour end point obtained and repeated to all the sample as above.

3.7 Cost analysis.

For the cost analysis cost of raw materials cost of labors and the cost of packaging
materials were used at the market prices in Sn Lanka at present

PX
Chapter 04.
4.1. Results and discussion.
4.1.1. Mushroom powder.
Oyster mushroom powder were prepared by dehydration and following grinding. At
present, oyster mushroom powder is available in many countries
(http://www.fuzing.com/vli/0004bl 230/Oyster-Mushroom-Extract-Powder-Pleurotus-
ostreatus). Ratio of fresh mushrooms to the yield of the powder was approximately 10:1.

4.1.2. Rehydrated flesh.

Faced difficulties while making paste owing to its too watery consistency as well as high
cost when comparing used of fresh flesh of mushrooms. Therefore both of the above are
economically and qualitatively not being accompanied with project objectives so gave up
the efforts.

4.1.3. The final product.

4.1.3.lQuality of the final product.
Only a few number of selected ingredients were used which are essential for the quality of
final product and also minimized the production cost. Sodium tri phosphate was selected
accordance with standard limits as well as to obtain binding, preservative action.

4.2 Collection of mushroom.

Only one brand of mushrooms was chosen. At the farm gate washed dry cleaned and well
packed raw material was collected so that attempt are not necessary for washing with water
if washed has to dry cleaned the raw material to maintain the quantity of water of the
formula because the formula has not been added the water.
Paste of mushroom was gained sending flesh of mushroom sending through the mincing
machine. It should be done at the later stage of the raw material preparation to avoid the
separation of the water part from the flesh. It's very important for protecting loss of
nutrients which are water soluble.

23
To obtain fme particle size of the paste mushrooms were sent twice through the mincing
machine. The fresh mushroom quality controlled must done in a proper ways because if not
it badly effect to the quality of final product.

Verity of oyster mushrooms was chosen because of its cheapness low wastage (minimized
preparation cost) and obtain paste of fme particle it was helped to maintained better texture
of the final product.

4.3 Processing of other ingredients.

As much as possible ingredients were collected one brand as well as in powder foam to
maintain better structure of the final product, to obtain higher flavorings from small
quantity of spices as well as minimized the quantity which used. Some ingredients such as
onion, cardamoms, cinnamon and garlic were chopped and finally ground to achieve the
above facts.

4. 4 Method used for processing mushroom sausages.

When mixing ingredients and paste of mushrooms salt and sodium tri phosphate should be
added early stages and shall allows them to proper binding properties to the final product.
By selecting sodium tri phosphate it affect are not only binding the ingredients it could
effect to maintain the longer the self life of the product due to its preservative action which
retard the growth of microorganisms.

4.5 Spices.
Chilly powder, pepper powder, turmeric powder were used in powder form it affect to
texture of final product. Some ingredients such as turmeric powder not only contribute to
the flavour profile of the final product but also it effect help to improve the colour of the
final product. All other spices which used here contribute to flavour profile and also give
some medicinal value to the final product e.g. Garlic. Some ingredients contribute flavour
profile of the final product. Carbohydrate product which was used here corn starch absorbs
excessive water that mean water holding capacity is higher. It gives good structural
properties to the final product and contributes to the slicing characteristics.

24
Salt is used to enhance the flavor, and to solubilize proteins in order to improve the binding
properties of the formulation. This enhances the product texture and improves water and fat
binding.

4.6. Processing steps.

While manufacturer mushroom base sausage must monitor and control the blending/mixing
process, since excessive mixing can cause the salts in the formulation to break down
excessive amounts of protein, or friction created by the blending process can increase the
product temperature and cause fats to partially render. Excessive handling also cuts protein
fibers too short. All of these problems could result in product quality defects so that it must
carefully be controlled.

The blending process must also obtain a uniform distribution of all ingredients within the
product formulation. For example, flavorings, salts, and other ingredients must be
consistently mixed throughout a sausage formulation. After the blending is complete, the
blended ingredients many be extruded into a casing. This process is called stuffmg. And
using icing nozzle stuff were inserted in to casing .then as much as possible remove the air
which enwrapped during mixing and filling it help to minimized microbial contamination,
fat oxidation, as well as harbor to the microorganism. The stuffed casings are then
separated into uniform segments of equal length in a process called linking. These
segments form the single sausage portions. The linking process is typically accomplished
by twisting and tied with threat. Then sausages were steamed it help to cooked the product
as well as it improved the texture by hardening. By steaming another important incident
occur it is sterilized the sausages by killing micro organisms. The next step is cooling the
sausage by running water it stop the over cooking and loss of nutrient by over cooking and
product should feel then should pack on plastic trays by wrapping with polythene and store
under cold room condition to retard the contamination and growth of microorganisms
Packaging is avoided any contaminating the product. However, the hygienic practice must
be followed all the steps as well as during all the process of manufacturing.

25
4.7. Result of microbiology.
4.7.1. Coliform.
During the four months of time duration there was no indication of contamination of
coliform.

4.7.2. Total plat count.

According to the SLS 516; part 1; 1991
Number of microorganisms (N) per grams of sausage samples
N=C/ (nl+o.1n2) d
Where YC= is the sum of colonies counted on all the dishes retained;

n1 = is the number of dishes retained in the first dilution;

n2 = is the number of dishes retained in the second dilution; and
d = is the dilution factors corresponding to the first dilution.
There were no colonies found in sausage samples which were tested.
So less than 1 * lid micro organisms per grams
Where d = is the dilution factor initial suspension.

4.8. Result and calculation of proximate analysis.

Average moisture percentage of sausage samples =70.475 5 (See table # 6 & Equation # 1)
Average ash percentage of sausage sample =3.4279 (See table #7 & Equation #2)
Average free fat percentage of sausage sample =3.1970 (See table # 8 & Equation # 3)
Average total fat percentage of sausage sample = 9.5780(See table # 9 & Equation # 4)
Average crude fibre percentage of sausage sample= 2.0896. (See table # 10 & Equation #
Average protein percentage of sausage sample = 2.84 (See table # 11 & Equation # 6)

26
Chapter 05
5.1. Conclusion.
The fmal product was assessed after keeping it for four month in cold room storage.
According to microbiological analysis and sensory evaluation it has been proven that the
ingredients used to prepare the fmal product were suitable and compatible with each other
in manufacturing the mushroom sausages. Similarly, the conditions applied to store the
sausage were suitable enough to keep the fmal product. According to quality characteristics
of fmal product it can be concluded that the basic objectives of the project were achieved.

5.2 Recommendation and suggestions.

The mushroom sausage satisfies the sensory requirements and has a shelf life of more than
four hours. However, the self life of the fmal product should continually be measured for
another two or more months of time as the product is still in very good condition.

Processing steps which were introduced can be changed and instead of applying non
cooked fresh flesh of mushroom there is a way of use pre cooked mushroom flesh with
spices mixture enhance the flavor profile of the final product.

27
References.
Baysal, E., Peket, H., Yalinkiliç, M.K. and Temiz, T. (2003) Cultivation of
oyster mushroom on waste paper with some added supplementary materials
Bioresource Technology, V. 89, 1, p 95-97

Bobek, P., Ozdin, L. and Kuniak, L. (1994) Mechanism of hypocholesterolemic

effect of oyster mushroom (Pleurotus ostreatus) in rats: reduction of cholesterol
absorption and increase of plasma cholesterol removal, Z Ern'-ihningswiss
33:44-50

Reddy, R (2000) Cultivation of oyster mushroom on en silkworm litter. mt J

Wild Silk moths silk V 5; Special Is sue ;p.263-265

Dried shiitake (Lentinulla edodes)and oyster (Pleurotus ostreatus) mushrooms

as a good source of nutrient. Acta Sci. Pol., Technol.Aliment. 6 (4), 135-142.Acta
Sci. PoL

Distance learning course manual, Association of food & drugs, version 01

September 1999.

Dr brian Imison & Dr daryl unthank a report for the rural industries research
and development cooperation.

A report for the Rural Industries Research and Development Corporation by Dr

Brian Imison and Mr Daryl Unthank April 2000 RIRDC Publication No 00/40
RIRDC Project No. DAV-97A © 2000 Rural Industries Research and
Development Corporation.

Madubashini Wijesinghe a thesis on analysis of added nitrite

& nitrate content in processed meat-sausages, Food Science

and Technology post graduate programs University of Sri

Jayewardenepura,Gangodawila,Nudegoda 2000.

P.AT.I. Perera, a thesis on improvement of jade manufacture

VI
 by improved curing salaya(Sardinella gibbosa) Food Science

and Technology post graduate programs University of Sri

Jayewardenepura,Gangodawila,Nudegoda 2002

Clifton,E. Meloan, Yashajahu Pomeranz, (2000), Food Analysis theory &

Practice, General applications and Chemical Composition, 3rd Edition, An
Aspen publication, pps.575 - 753.

Kirk Ronald S. and Sawyer Ranold, (1999), Pearson's composoion and analysis
of food, Addisan Wesley Longman.Inc.,lOOpp.

Neilsen Suzanne 5, (1998), Food analysis,2nd Edition, Aspen publishers, 127-

217pp.

Potter Norman,N. and Hotchiclogiss Joseph, H.(1980), Food sciences, 5th

Edition, CBS publishers and distributors, 50-60pp.

Sathe, A.Y.,(1999), A first course in food Analysis, New Age International

Publishers ltd. 100-120pp.

Clifton,E. Meloan, Yashajahu Pomeranz, (2000), Food Analysis theory &

Practice, General applications and Chemical Composition, 3rd Edition, An
Aspen publication, pps.575 - 753.

Milk testing, dairy Science and technology, University of Guelph,

http://www.foodsci.uoguelph

www.mc.maricopa.edu/—johnson/imvic.html.

www.austincc.edu/microbugz/html/imvic.htinl

0 www.fao.org/docrep/003/x6556e/x6556e07.htm.
. www.keziefoods.co.uk.

www.wikipedia.org.
www.advancedbionutritional.com.

www.ffickr.com.

www.fuzing.com/vli/0016104b1230/Oyster-Mushroom-Extract-Powder-
Pleurotus-ostreatus

VIII
Table #7 Results & Calculations.
Sample# 1 2 3

Weight of dish with lid(rnlg) 44.6397 47.53904.3523

Weight of sample (g) 5.4566 5.4450 5.4345

Weight of dish with lid and sample before drying(m2g) 50.0963 59840 68

Weight of dish with lid and sample after drying(m3g) 46.2507 49.1452 45.9583

Moisture c)ntent % 70.4765 70.5016 1 70.4486

Equation 01
Moisture % = Weight loss
\Veihl o the sample

= x 100

m2-ml

Table 98: Results & Calculations of Ash

Sample# 1 2 3

Weight of crucible (g) 34.2005 20.5700 18.0173

Weight of sample (g) 5.6 143 ~

5. 752 5.5822

Weight of crucible& ash (g) 34.4014 .7658 115

8. 9 4

% of ash 3.58 3.5126 3.1911

Equation 2
Ash% = Weight of ash x 100
Weight of sample
X 100
I-ni

Ash % (on dry basis) im-m2 xlOOx 100

(1 00-%rnoisture)

Average ash content (on dry basis) = 3.4279 %

Ix
Table 49 Results & Calculations of free fat:
Sample# 1 2 3
Weight of ground sample 5.0026 5.1121 5.1540
Weight of round bottom flask ± chips(m2)g 159.214 159.202 159.012
Weight of round bottom flask+chips+fat (Afler drying)(11-13)9 159.3742 159.3664 159.1262

%of free fat 3.2013 3.21 3.1767

Equation 3

Crude fat content = (Weight of flask +fat +chips) - (Weight of flask+chips) x 100
of the sample % weight of the sample

= (ni3) g(m2) g x 100

(m)g

Table # 10 Results & Calculations of total fat:

Sample# 1 2 3
Weight of flask 104.175 108.574 60.111
3 1 3
Weight of ground sample 1.8759 2.2769 2.2763
J
Weight of flask & fat extract 104.723 108.792 60.326
6
% of total fat 9.6983 9.6001 9.4356

Equation 4
% Total fat = weight of fat x 100
Weight of sample

x
 Table #11 Results and Calculations of crude fibre:
iple 1 2 3
Weight of sample 2.1138 2.1717 2.1073
Weight of crucible 30.6546 31.5673 1 30.3215
Weight of crucible ± filter paper 31.3547 1 32.2674 31 .0234
Weight of crucible + filter paper + residue before ashing 31 .4045 32.3161 3 I .0687
Weight of crucible + filter paper + residue afterashing 30.6580 31.5706 30.325

[
~o Crude Fiber 2.1950 2.0905 1.9835

Equation 5
% Crude Fiber = Loss in weight on incineration x 100
Weight of sample

= Wt of residue - Wt of Ash x 100

Wt of sample

Table # 12 Results & calculation of protein:

Sarnple# blank 1 2 3
Weight of sample(g) *
1.0723 1.0582 1.0180
Volume of HCJ (ml) *
17.40 17.20 16.50
Nitrogen % *
0.4543 0.4551 0.4538
Protein % (x 6.25) *
2.8393 2.8443 2.8364
Average % of protein *
2.84

Equation 6
Nitrogen % = (Sample titre - Blank titre) x( Molaritv of FICI x 14 xlOO)
Wt of the sample taken x 1000

Protein % = Nitrogen % x 6.25

xlr
Appendix.

Ballot paper for sensory analysis.

Name. ..................................... Time.........................

7I1ke very much,

6Iike moderately,
5=like slightly.
4=neither like nor dislike,
..)dislike slightly,

2= dislike moderately,
1= dislike very much,

Sample code Colour appercnce Taste TextureOverall

acceptability

Comments;

Thank you.

XII