Study on fungi associated with spoilage of bread and its

inhibition by lactic acid bacteria in Bannu

KPK, Pakistan.

BS ZOOLOGY

BY

Quratul-ain

Department of Zoology

Govt. Girls College Mandan, Bannu

KP, Pakistan

2018-2022

Study on fungi associated with spoilage of bread and

its inhibition by lactic acid bacteria in Bannu KPK, Pakistan

 Certificate of Approval:

This is to certify that the Department of Zoology, Faculty of Biological Science, Govt.
Girls Degree College Mandan Bannu KP, Pakistan accepts the dissertation entitled
“Study on fungi associated with spoilage of bread and its inhibition by lactic acid
bacteria, in Bannu KPK, Pakistan" submitted by Quratul-ain in its present form as
satisfying the dissertation requirement for Degree of BS (Hons) in Zoology.

Supervisor:

External:

Principal:

Date:

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 Dedication:

I would like to dedicate my work to my beloved Parents

Mr. and Mrs. Zafrullah khan

Who’s Love, Encouragement, Prayers, and Support brought me to this wonderful

success.

They have been my constant source of inspiration.

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 Declaration:

I, Quratul-Ain, hereby declare that work in this thesis titled “Study on fungi associated
with spoilage of bread and its inhibition by lactic acid bacteria in Bannu KPK,
Pakistan” has been carried out by me in the Department of Zoology. The information
derived from the literature has been duly acknowledged in the text and a list of references
is provided. No part of this thesis was previously presented for another degree or diploma
at this or any other institution.

Signature:

Quratul-Ain

BS Zoology

Department of Zoology

Govt. Girls Degree College Mandan, Bannu

Session: 2018-2022

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 Acknowledgment:

In the name of Allah, the Most Gracious and the Most Merciful Alhamdulillah, Within
the title of Probably the Most Thoughtful Allah and also the Merciful Alhamdulillah, I
reward and appreciate Him, consult Him for His aid and Forgiveness, and that I find
sanctuary in Allah from the mischief’s of our actions and also the evils of our spirits. He
whom Allah guides will not be misled, and he whom Allah misleads will never have a
guide. I state that there's no deity but Allah alone, with no companions, and that
Muhammad (S.A.W) is His 'Abd (worshiper) and Messenger. First of all, I appreciate
Allah (Subhana WA Taala) for bestowing me with wellness, persistence, and
understanding to accomplish this thesis. Allah had given me the power for this chance
and also the persistence to accomplish my dissertation after the challenges and hard work.

I am extremely grateful to my parents Mr. and Mrs. Zafrullah khan for their love,
caring, support, prayer, and sacrifices for educating and preparing me for the future. I am
also thankful to my grandmother for her prayers.

Very sincere and greatest thanks to my respected, friendly, and worthy honorable
Principal Musarrat Shaheen who tends out to be an inspiration throughout my study
period.

Special appreciation goes to my supervisor, Ma’am Huma Arif, for her supervision and
constant support. Her invaluable help of constructive comments and suggestions
throughout the experimental and thesis works have contributed to the success of this
research.

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I owe my greatest thanks to my respected, friendly advisors Ma’am Zakia Minhas,
Department of Zoology, whose valuable guidance and keen interest help me a lot to
fulfill my work.
My completion of this project could not have been accomplished without the support of
my friends, Naila, Sobia Owais, and Aimen Rehman and all my friends of
microbiology.

Lastly, I am thankful to all my Family, Friends, my entire teachers of the school, college,
and lecturers of the Department of Zoology, who motivated and fostered me to achieve
greater heights in knowledge.

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 Table of Contents

Certificate of Approval: .................................................................................................. i

Dedication:....................................................................................................................... ii
Declaration: .................................................................................................................... iii

Acknowledgment: .......................................................................................................... iv
Table of Contents .............................................................................................................. vi

List of Graph: ................................................................................................................. ix

List of Figures:................................................................................................................. x

List of Tables .................................................................................................................. xi

Abstract: ........................................................................................................................ xii
CHAPTER 1 ....................................................................................................................... 1

INTRODUCTION: ......................................................................................................... 1
Bread: ............................................................................................................................... 2

Types of bread: ................................................................................................................ 3

White bread: .................................................................................................................... 3

Brown bread: ................................................................................................................... 3
Whole meal bread: .......................................................................................................... 3

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 Rye bread: ........................................................................................................................ 4
Nutritional significance: ................................................................................................. 4

Calcium ............................................................................................................................ 4

Fiber ................................................................................................................................. 4
Protein .............................................................................................................................. 5
Iron ................................................................................................................................... 5

Vitamins & Other Minerals ........................................................................................... 5

Energy .............................................................................................................................. 5
Fat ..................................................................................................................................... 5
Sugar ................................................................................................................................ 5

Preparation of bread: ..................................................................................................... 6

Bread mould: ................................................................................................................... 7
Genus Rhizopus:.............................................................................................................. 7

Rhizopus stolonifer: ......................................................................................................... 8

Ecology ............................................................................................................................. 8
Physiology ........................................................................................................................ 9
Lactic acid bacteria: ....................................................................................................... 9

CHAPTER 2 ..................................................................................................................... 11

LITERATURE REVIEW: ........................................................................................... 11

CHAPTER 3 ..................................................................................................................... 17
Material and Method: ................................................................................................... 17

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 Material: ........................................................................................................................ 17
Methodology:................................................................................................................. 18

Sample collection: ......................................................................................................... 18

Sterilization: .................................................................................................................. 20
Media preparation: ....................................................................................................... 20
Media pouring: .............................................................................................................. 21

Streaking: ...................................................................................................................... 22

Incubation: .................................................................................................................... 22
Microscopic examination: ............................................................................................ 23
Dual culture overlay assay method: ............................................................................ 24

CHAPTER 4 ..................................................................................................................... 25
Results:........................................................................................................................... 25
Culture examination: .................................................................................................... 27
Microscopic examination: ............................................................................................ 27
Fungus inhibition: ......................................................................................................... 29
Discussion: ..................................................................................................................... 31
Conclusion: .................................................................................................................... 32

Future Prospective: ....................................................................................................... 33

Recommendations: ....................................................................................................... 34
REFERENCES: ............................................................................................................ 35

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 List of Graph:

S.No Title Page#

1 Fungal growth on the basis of scale 26

2 Fungus inhibition 30

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 List of Figures:

S.NO. Title Page #

1 Figure 1 freshly baked and spoiled bread samples 19

2 Figure 2 cleaning of working surface 20

3 Figure 3 Media preparation 21

4 Figure 4 Media pouring 21

5 Figure 5 streaking 22

6 Figure 6 Incubator 23

7 Figure 7 Microscopic examination 23

8 Figure 8 Agar overlay 24

9 Figure 9 culture examination 27

10 Figure 10 Microscopic examination 28

11 Figure 11 Fungus inhibition 29

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 List of Tables

S.No Title Page#

1 Table 1 Fungal growth on the basis of scale 25

2 Table 2 Rhizopus inhibition by LAB 29

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 Abstract:

Bread is one of the staple foods in the world and it is recognized as a semi-perishable
commodity. Bread is a good source of carbohydrates and nutrients such as magnesium,
iron, selenium, B vitamins, and dietary fiber. Bread is easily damaged, especially due to
fungi attack. Bread mould is a kind of fungus that is commonly found on bread surfaces.
We study bread molds about Rhizopus stolonifer appeared on bread and its inhibition by
Lactic acid bacteria from homemade curd. Rhizopus stolonifer is the most common type
of household mold grows primarily on bread and actually the most prevalent type of mold
in the world. Their spores spread through the air and grows quicker in a moist or wet
environment, particularly within a range of 15 degrees to 30 degrees Celsius. Lactic acid
bacteria are characterized as gram-positive, usually non-motile and non-sporulating that
produces lactic acid as a major product of fermentative metabolism. Bread can be
protected from spoilage microorganisms by destroying the spores which contaminate the
products, using Lactic acid bacteria with broad antifungal spectrum. Total 71 samples
were cultured on potato dextrose agar for fungal growth and nutrient agar for bacterial
growth. Rhizopus stolonifer was examined under microscope based on its morphological
characteristics. By dual culture overlay method fungal inhibition was observed by
measuring the zone of inhibition due to acidic conditions that Lactic acid bacteria create,
converting carbohydrates to organic acids (lactic acid and acetic acids) in the food during
their development.

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CHAPTER 1

INTRODUCTION:

Bread is a group of carbohydrate foods that should be consumed by about one-third of the
diet. Bread like pasta, potatoes, rice, and other cereals, is the best food source of energy.
These foods also contain B complex vitamins to release energy from food and maintain a
healthy digestive and nervous system (Marshal 2005).

Bread is made from flour that is spread with yeast and baked. Bread is easily damaged,
especially due to fungi attack. The types of fungi that often contaminate food are mold
and yeast, but the more dominant ones are molds.

Molds are able to grow on all kinds of food: cereals, meat, milk, fruit, vegetables, nuts,
fats and products of these. The mold growth may result in several kinds of food spoilage:
off-flavors, toxins, discoloration, rotting and formation of pathogenic or allergenic
propagules (Wareing 2012).

Fungi are microbes consisting of more than one cell in the form of fine threads called
hyphae, a collection of hyphae called mycelium, reproducing by spores or dividing (SNI
7388; 2009). As a result of growing fungi on bread so that bread cannot be reconsumed.

According to the previous studies (Banwart, 2004) bread molds like Mucor and Rhizopus
are found to grow first during bread spoilage. This is followed by some other fungi like
Aspergillus, Penicillium and Fusarium sp. In this study we are concerned with Rhizopus.

In addition to the studies, the fast growth of fungi on bread often causes bakers to
experience losses due to the low storage capacity of bread (BSN 2009).

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Packaged bread can be protected from spoilage microorganisms by destroying the spores
which contaminate the products, using Lactic acid bacteria with broad antifungal
spectrum due to acidic conditions that they create, converting carbohydrates to organic
acids (lactic acid and acetic acids) in the food during their development. (Goyal et al.,
2012) Lactic acid bacteria are characterized as gram-positive, usually non-motile, non-
sporulating that produces lactic acid as a major product of fermentative metabolism.

Bread:

Bread or in Greek ‘‘artos’’ was considered in the past as the most important human good
after health and water. The origin of the term ‘‘artos’’ is ancient Greek: it comes from the
verb “ararisko” which means connect, match, put together or ‘‘artio’’ which means
brew, and prepare.

The modern term ―bread‖ derived from the ancient verb “psoo” meaning rub and it is
diminutive of the word “psomys” (→ bread = little bit, bite).

It is the main food in Europe but also in the cultures of America, Middle East and North
Africa, unlike East Asia, where the main food is rice. The bread, therefore, a food with
awesome variety of flavors and nutrients remains the basis of our daily diet. However, it
is accused of contributing to weight gain etc.

Bread wheat is the most common grain used for the preparation of bread, which makes
the largest single contribution to the world's food supply of any food.

Bread is also made from the flour of other wheat species (including spelt, emmer, einkorn
and kamut). Non-wheat cereals including rye, barley, maize (corn), oats, sorghum, millet
and rice have been used to make bread, but, with the exception of rye, usually in
combination with wheat flour as they have less gluten.

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 Types of bread:

Bread is used as food in the developed and developing countries. East Asian societies
typically prefer wheat noodles, steamed bread, or rice. Bread is often made from wheat-
flour dough that is cultured with yeast, allowed to rise, and finally baked in an oven.

Owing to the high levels of gluten (which give the dough sponginess and elasticity),
common wheat (also known as bread wheat) is the most used grains for the preparation of
bread. It is also made from the flour of other grains species (including sorghum, rye,
barley, maize (or corn) and oats (Wikipedia, 2008). Although common wheat is the best
suited for making highly-risen white bread, other wheat species are capable of giving
good black bread.

White bread:

White bread is made from flour containing only the central core of the grain (endosperm).
It is the most commonly eaten bread.

Brown bread:

Brown bread is made with endosperm and 10% bran (whole wheat). It can also be
referred to as white bread with added coloring to make it brown.

Whole meal bread:

Whole meal bread contains the whole of the wheat grain (endosperm and bran). It is also
referred to as "whole wheat" bread, especially in North America. It is high in fiber than
many common types of bread and is often darker in color and stronger in flavor.

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 Rye bread:
In Scandinavia, Germany, Finland, the Baltic States, and Russia, rye is a popular type of
bread. Rye bread is made with flour from rye grain of variable levels. It is higher in fiber
than many common types of bread and is often darker in color.

Bread rolls in many varieties are an important daily food for city dwellers. In Britain
there is a wide variety of traditional bread often baked in a rectangular tin. In South Asia
(India, Pakistan and Bangladesh) some types of flat breads are commonly used.

A thin flat bread which is fried rather than baked and puffs up while cooked. Paratha is a
type of bread baked in clay oven and is rarely prepared at home. White and brown bread
are also very common.

Nutritional significance:

Nutritionally, bread is categorized as a source of grains in the food pyramid. Further, it is

a good source of carbohydrates and nutrients such as magnesium, iron, selenium, B
vitamins, and dietary fiber.

Calcium
White bread is fortified with calcium and four medium slices per day would provide over
30% of the recommended daily intake of calcium which we need every day to maintain
healthy bones and teeth.

Fiber

Bread, especially whole meal, is an important source of dietary fiber which helps to keep
our digestive system healthy, helps control blood sugar and cholesterol levels and makes
us feel fuller for longer.

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 Protein

Bread is a low fat source of protein which is required by our bodies for growth, renewal
and repair.

Iron

White bread is fortified with iron. Iron is important for energy and concentration, a
healthy immune system and healthy blood.

Vitamins & Other Minerals

Bread contains a wide range of vitamins and minerals including B group vitamins
thiamine (B1), Niacin (B3) which is important for releasing energy from food and
maintaining healthy skin, eyes and nails. It contains the B vitamin Folate (Folic Acid)
which is important for pregnancy as it can help to prevent neural tube defects such as
spina bifida.

Energy

Bread is relatively low in calories. An average medium slice of white bread contains 77
calories, brown contains 72 calories and whole meal contains 79 calories.

Fat

Bread is a low fat food. An average medium slice of white bread contains 0.6g of fat,
brown bread contains 0.7g and whole meal contains 0.9g. Just be careful with what you
put on it and stick to healthy options for spreads and toppings.

Sugar

Most bread is low in sugar which is important for healthy teeth and maintaining a healthy
weight.

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 Preparation of bread:

The basic ingredients in bread includes: flour to give strength and structure of products
by starch and protein contained in the flour; water as a solvent to act with flour to form
dough; yeast to act on natural sugars in the flour to generate carbon dioxide, and to make
the dough rise through fermentation. The activity of yeast is strongly associated with
dough fermentation; salt not only to contribute to flavor but also to strengthen the gluten
and control the action of yeast for loaf volume.

Apart from these minimal necessities small amounts of extra ingredients are added to
enhance dough performance during processing or to improve the quality of finished
bread. As such, sugar is added to provide nutrients to yeast in the early stages of
fermentation. Fat or milk derivatives are also added to improve the quality of the produce
by contributing softness, moistness and addition flavor and texture. In addition, other
functional components including additives and preservatives might be used in flour or
dough to improve baking quality of bread and yeast-raised fine bakers’ wares (Williams
and Pullen, 2007).

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 Bread mould:

Bread mould is a kind of fungus that is commonly found on bread surfaces. It takes food
and nutrients from the bread and causes damage to the surface where it lives. It causes a
bad taste to the bread. But the mould has a place in the industry where it serves as a
decomposer that can decompose decayed plants and animal. Bread mould has a very
simple life cycle. It appears on the bread surface as a wind-blown spore. With adequate
moisture and nutrients from the bread, this spore sprouts and grows hair like structures on
the bread surface. Once the mould attains a particular growth, it starts producing fruiting
spores' structures. These structures sometimes called conidia contain spores that are
blown by wind and spread to other bread surfaces.

Bread mould is found in different types, species, shapes, and color, some of the common
bread mould is Penicillium, Aspergillus, Rhizopus and Fusarium. Here we are studying
about Rhizopus appeared on bread.

Genus Rhizopus:

It is a cosmopolitan genus of some 10 species of filamentous fungi in the family

Rhizopodaceae (formerly Mucoraceae), in the order Mucorales. Several species,
including Rhizopus stolonifer (the common bread mold), have industrial importance, and
a number are responsible for diseases in plants and animals.

The majority of Rhizopus species is saprobic (decomposers) and feed on a variety of dead
organic matter, though some species is parasitic or pathogenic.

Rhizopus fungi are characterized by a body of branching mycelia composed of three

types of hyphae: stolons, rhizoids, and usually unbranching sporangiophores. The black

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sporangia at the tips of the sporangiophores are rounded and produce numerous non-
motile multinucleate spores for asexual reproduction.

Rhizopus can reproduce sexually when two compatible and physiologically distinct
mycelia are present. The rapidly growing colonies fade from white to dark as they
produce spores and are similar to cotton candy (also called candy floss or fairy floss) in
texture.

Rhizopus stolonifer:

The most common type of household mold grows primarily on bread, but also lives on
many other foods. This type of mold, Rhizopus stolonifer, is actually the most prevalent
type of mold in the world.

It is more commonly known as black bread mold, although it more likely to appear green,
grey, or even white. Over time it will darken and occasionally become black on certain
hosts. Although sharing a similar name as the dangerous household black mold
(Stachybotrys), it comes from a different phylum of mold and is significantly less
dangerous.

Ecology

Rhizopus stolonifer is of the Fungi Kingdom, the Mucorales Order, and Mucoraceae
Family. It is most commonly found on bread, but will occur on crop type vegetables such
as papayas and sweet potatoes. In vegetables like, this it will cause soft rot along with the
usual appearance of green color and white fuzz.

The mold itself is a mass of mycelium which is comprised of multinucleate and when the
spores are released the mycelium is produced in greater quantities via germination. To
survive, black bread mold needs sugar or starch for its source of carbon substances for

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 food. Due to these reasons, healthier breads like whole wheat will develop mold more
quickly as opposed to white bread which lacks the nutrients for bacteria to thrive.

Physiology

The spores spread through the air and grows quicker in a moist or wet environment,
particularly within a range of 15 degrees to 30 degrees Celsius (59-86 Fahrenheit).

Rhizopus stolonifer is a heterothallic species meaning it produces sexually but only when
opposing mating types (+ and -) are paired. Zygospores are produced when this sexual
contact occurs and they then sporulate to create a sporangiophore. This contains a
sporangiophore that contains both + and - haploid species.

Lactic acid bacteria:

Lactic acid bacteria play an important role in the dairy and meat fermentation process and
have a great influence on the quality and preservation of the end products. Lactic acid
bacteria are characterized as gram-positive, usually non-motile and non-sporulating that
produces lactic acid as a major product of fermentative metabolism. The preservative effect
of lactic acid bacteria during the manufacture and subsequent storage of fermented foods is
mainly due to acidic conditions that they create, converting carbohydrates to organic
acids (lactic acid and acetic acids) in the food during their development. (Goyal et al.,
2012)

Genus Lactobacillus:

Lactobacillales is one of the diverse and phylogenetically heterogeneous orders of lactic

acid-producing bacteria that include the type genus Lactobacillus, as well as the genera

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Facklamia, Granulicatella, Leuconostoc, Pediococcus, and Streptococcus. They utilize
carbohydrates fermentatively and produce lactic acid as a major end-product.

Lactobacillus sp is facultative anaerobic, catalase-negative, Gram-positive, non-spore-

forming rods that often grow better under microaerophilic conditions. Lactobacilli grow
on a variety of other media including MRS (Man, Rogosa, and Sharpe) agar where they
appear as white, usually mucoid colonies. Identification of Lactobacillus species is by
molecular means (16S rRNA genes) as phenotypic identification is generally unreliable.
(Goldstein, Tyrrell, & Citron, 2015)

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CHAPTER 2

LITERATURE REVIEW:

Bread is the major component in the overall consumption of grains, particularly whole
grains. There is an increasing variety of organic, ―natural,‖ and conventional bread—
white, wheat, whole wheat, sprouted wheat, and gluten-free. Bakery products and cereals
are a valuable source of nutrients in our diet providing us with most of our food calories
and approximately half of our protein requirements. Cereals have been a basic food of
man since prehistoric times and were consumed long before bread making was
developed. Variety breads and other bakery products have increased in sales volume
within the past decades. The nutrients in bakery products are carbohydrates, proteins,
lipids, vitamins and minerals (Chavan and Kadam 1993).

Bread is one of the staple foods in the world and it is recognized as a semi-perishable
commodity. Usually the spoilage of bread is due to improper storage. The fungi
associated with the spoilage of bread were studied. Mold spoilage of bread is due to post
processing contamination. Twenty bread samples were collected from different shops in
Jaffna. The fungi involved during spoilage were isolated and identified based on the
cultural and morphological characteristics using the standard keys available. Initially the
molds namely Mucor sp and Rhizopus sp were found to be the cause of bread spoilage.
This was followed by Aspergillus sp and Penicillium sp. In this study Rhizopus sp was
found to be the most common fungus during the spoilage of bread.

Bread provides an ideal environment for fungal growth regarding nutrient‚ moisture and
temperature. Such contamination becomes visible to the eye ranging from white ‚ green
and black colors in late stages. These fungal contaminants may harmfully affect the
consumers due to their effect on bread taste ‚ smell‚ odor and other substances secreted in

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the bread. So ‚the study is aiming to detect different fungal their incidence during the
contaminants occurring in bread and handling of the bread. Blotter and agar plate
methods were used for fungal detection. Bread samples were brought from bakeries ‚
house‚ restaurants and from the open planks ready for sale. The results showed that all
collected samples were contaminated with fungi but to different extent. Aspergillus niger,
A. flavous ‚ Rhizopus sp and Penicillium sp were detected on contaminated bread. Blotter
method yielded more fungal growth comparing to agar plate method. As for the keeping
methods ‚ it was found that keeping the bread in plastic bags is better than the plastic
buckets and the refrigerator. More studies are needed in this area as the bakeries differ in
their types of buildings and care for the cleanness and hygiene. In addition to that‚ the
mycotoxins are hazardous poisons that need to be studied.

Bread is a highly perishable product. The three most common forms of bread
deterioration are staling, moisture loss and microbial spoilage (Seiler, 1984, Legan,
1993). Losses due to mould spoilage are difficult to quantify. However, a conservative
estimate of 1% would result in losses of 20 million pounds in the U.K. alone every year.
Across Europe, the economic costs would be in the region of 5-10 times greater. The
reason why moulds are important spoilage organisms in bread is because this food matrix
has a relatively high moisture content (= 0.94-0.97 water activity (aw)) with a pH of
about 6. These environmental conditions are conducive to germination and growth of any
moulds contaminating the bread during or post-production. The bread most prone to
spoilage by moulds is sliced, prepacked and wrapped bread. This type of product
provides moist cut surfaces for moulds to grow on and wrapping prevents moisture loss
allowing a humid atmosphere around the loaf. More than 90% of contamination of bread
with moulds occurs during cooling, slicing or wrapping operations (Spicher, 1984). Prior
to this stage the heating regimes employed in baking mean that most contaminants are
eliminated (Legan, 1993; Roesler and Ballenger, 1996). Thus contamination is
predominantly by fungal spores being deposited from the bakery environment, from flour
dust and by introduction from the outside atmosphere. Many different filamentous mould
species have been implicated in mould spoilage of bread including Penicillium, Rhizopus

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species, Cladosporium species, Mucorales and Neurospora (Legan, 1993). Tolerance to
wide range of environmental conditions, and their predominantly mycelial growth habit
enable them to colonies food products rapidly producing a battery of enzymes to utilize
the food matrix. This study aimed to verify the main fungal species involved in the
deterioration of different types of bread and to identify the possible sources of
contamination of these products. Samples of raw materials (n = 127), environmental air
(n = 50) and moldy bread (n = 90) were analyzed. Rhizopus stolonifer, Wallemia sebi,
and Penicillium roqueforti were the predominant species in the raw materials and were
isolated in samples of wheat flour, in two-thirds of the samples of rye and 62.5% of the
wheat flour Rhizopus stolonifer was isolated from all types of moldy bread analyzed and
also present in samples of moldy wheat and rye bread. These species were also recovered
during air sampling from baking industry facilities (cooling and slice and package areas),
which may be crucial for product contamination after baking. Hygienic measures to
reduce airborne contamination during the cooling and packaging of food should be taken
to prevent the early deterioration of bread.

Mouldiness is one of the most common microbiological defects, found in bakery

industry. It is estimated that approximately 1-5% of the bread production goes wrong due
to fungi activity. Referring to bread, mold contamination determines not only changes in
color, taste, but also loss of the food quality as a result of possible formation of
mycotoxins. Mouldiness is caused by external contamination of bread after baking,
because the existing spores in flour during a normal technological process don’t have any
multiplication conditions, and during baking they are destroyed. Bread contamination
with molds, may occur in the following steps: the transportation of bread; during cooling
and storage; while cutting and packing (optional operation). Propagation of mold spores
and contamination of bread can take place indirectly by air in the storage room (the air)
or by direct contact with boxes, crates, hauling equipment etc. Among the molds that
frequently contaminate bread during storage, there are the following common species and

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genera: Alternaria, Aspergillus, Cladosporium, Geotrichum candidum, Mucor,
Neuropspora sitophila, Penicillium, Rhizopus stolonifer, Thamnidium elegans. By the
development of other fungi superficial pigmentation are produced, which only affects the
commercial appearance of bread. Storage of bread under conditions of low humidity
retards mold growth.

In addition to the economic losses associated with bakery products, another concern is the
possibility of mycotoxins production. Eurotium species are usually the first fungi to
colonize improperly water allowing other species, Rhizopus and Penicillium, which can
produce toxins to thrive. Losses of bakery products due to mold spoilage vary between 1–
5 percent depending on seasons, type of products and methods of processing (Abellana et
al. 1997). Since, filamentous fungi are more easily recognized than yeast, they generate
the majority of complaints. Longer shelf life enables a greater variety of products to be
kept in store and in the home (Abellana et al. 2000). Spoilage from microbial growth
causes economic loss for both manufacturers and consumers. These losses could be due
to many individual cases such as packaging, sanitary practice in manufacturing, storage
conditions and product turnover. Needham et al. (2005) tested the microbial spoilage
caused by bacteria, yeast and fungi and enzymic spoilage caused by lipoxygenase can be
differentiated from one another and from unspoiled bread analogues after 48hrs using
cluster analysis, prior to signs of visible spoilage.

Twenty one bacterial isolates were obtained from the normal habitats of lactic acid
bacteria (LAB). They were isolated from women milk, sheep milk, yoghurt, pasterilized
milk and fermented food" pickles". Screening of all isolates of LAB for antimicrobial
activity on MRS medium revealed that the highest antimicrobial activities were against
Rhizopus stolonifer. The isolate W9 was the most active isolate and it was characterized
and identified through physiological and biochemical tests, in addition to examination
with light and scanning electron microscope as Lactobacillus bulgaricus W9. The
maximum antimicrobial activity of the selected isolate W9 was achieved using MRS

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medium after 48 hours of incubation in aerobic conditions at 30ᵒC and pH 6 of the culture
medium. The antimicrobial agent was extracted and crude extract affected the growth of a
number fungi. The antimicrobial compound decreased fungal growth and mycotoxins
production. Thus, they can be used as food bio preservative.

One potent lactic acid bacterial strain C14 with strong antifungal activity was isolated
from homemade curd. Based on morphological as well as biochemical characters and 16S
rDNA sequence homology the strain was identified as Lactobacillus fermentum. It
displayed a wide antimicrobial spectrum against both Gram-positive and Gram-negative
pathogenic bacteria, and also against number of food spoilage, plant and human
pathogenic fungi. The cell free supernatant (CFS) of the strain C14 was also effective
against the fungi tested. Inhibition of radial growth of Penicillium digitatum,
Trichophyton rubrum and Mucor sp. was noticed in the presence of CFS of C14 even at
low concentration (1%). More than 94.3 ± 1.6% and 91.5 ± 2.2% inhibition of conidial
germination of P. digitatum and Mucor sp. were noticed in the presence of 10-fold-
concentrated CFS of C14. Massive deformation of the fungal mycelia was observed by
SEM studies, and losses of cellular proteins and DNA are also evident upon its treatment
with C14. HPLC analysis revealed the presence of phenyl lactic acid, lactic acid along
with some unidentified compounds in the antifungal extract. Challenge experiment
showed immense potential of the strain C14 in preventing the spoilage of bread samples
caused by Mucor sp. and Bacillus subtilis. The bread samples remained fresh upto 25
days even after inoculation with Mucor sp. (3.7 × 104 spores /ml) and B. subtilis (4.6 ×
104 CFU /ml). Along with the antifungal properties, the isolated lactic acid bacterial
strain also showed very good antioxidant activities. Unchanged level of liver enzymes
serum glutamic pyruvic transaminase and serum glutamic oxaloacetic transaminase in
albino mice upon feeding with C14 also suggested non-toxic nature of the bacterial
isolate.

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Lactic acid bacteria (LAB) belonging to Gram positive group, are generally recognized as
safe (GRAS) and usually play an important role in food and feed fermentations as well as
in their preservations. Lactobacillus is one of the largest genus in this category with
almost 80 species that are widely used in different food products including sauerkraut,
beer, pickle, wine, juices, cheese, yogurt and sausage. LAB are also used as starter
cultures for the production of dairy, meat and vegetable fermentation products. In
addition to their fermentation properties, a large number of LAB strains exhibit very good
antifungal and antibacterial activities. Therefore, they are used as bio preservatives.
Lactobacillus inhibits the growth of Rhizopus production.
According to the Center for Science in the Public Interest (CSPI) various chemical
preservatives cause serious health hazards to the mammalian system. Excess intake of
sodium nitrate may cause tumor and cancer, salt form of sodium bicarbonate or baking
soda increases blood pressure. Chemical preservatives like potassium bisulfate and
potassium meta bisulfate exhibit allergic symptoms. On the other hand, LAB are quite
safer and merely have any side effects on mammalian system.

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CHAPTER 3

Material and Method:

Material:

o Protective gloves
o Mask
o Slides
o Slide covers
o Distilled water
o Sterilized Conical flask
o Digital Balance
o Filter Paper
o Digital Magnetic Stirrer
o Petri Dishes/Agar plates
o Cotton Plug
o Paper Tape
o A Marker
o Autoclave
o Spirit lamp
o Incubator
o Ethanol/Spirit
o Micropipette
o Biosafety Cabinet
o Inoculation loop
o Laminar flow
o Microscope

o Forceps

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 o Spoiled bread samples
o Potato dextrose agar
o Nutrient agar
o Homemade curd

Methodology:

 Sample collection
 Sterilization
 Media preparation
 Media pouring
 Streaking
 Incubation
 Microscopic examination
 Dual culture overlay assay method

Sample collection:

Bread was baked at home from 3 types of flour, purchased from local shops in Bannu.
Wheat, maize and barley flour was used for bread baking. Bread samples were then
packed in plastic bags and leave for 5-7 days. Molds were appeared on bread samples in
the form of blackish spores. Homemade curd samples were also taken to obtain LAB.
Afterward, all samples were taken to Government Girls Post Graduate College Mandan,
Bannu for further analysis.

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 Wheat Maize Barley

Freshly baked bread

Wrapped in plastic

Fungal attacked

Figure 1 freshly baked and spoiled bread samples

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 Sterilization:

We washed all the petri dishes and a conical flask. These petri dishes were wrapped and
then put inside the autoclave for sterilization at 121°C for 15 minutes. At that time we
clean our working surface using cotton dipped in ethanol.

Figure 2 cleaning of working surface

Media preparation:

Potato Dextrose Agar (PDA) which is a common medium to grow fungi was used in this
study. We weigh 39g of PDA and dissolved it in 1000ml of distilled water in a glass flask
and shake the flask well using a shaker until the entire gradient is completely dissolved in
water.

We also prepared nutrient agar for lactic acid bacteria growth for which we dissolved 7g
of agar in 250ml of distilled water in a glass flask.

Closed the mouth of the flask with aluminum foil and placed in autoclave. Then this
medium was autoclaved at 121 ˚C for 15 minutes along with petri dishes.

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 Figure 3 Media preparation

Media pouring:

After steamed sterilization, we brought the sterilized media and Petri plates to the laminar
hood. Inside the laminar hood, we turned on the spirit lamp and opened the plates from
sterilized packing, and remove the aluminum foil from the flask. Shake the media in the
flask well so that all ingredients were mixed well. We were placing tip of the flask on
spirit lamp each time when we pour the media. Pour the sterilized media into sterilized
Petri plates inside the laminar hood in equal amounts. After pouring the media into plates
in equal amounts and we leaved it for a few minutes so that the media is properly
solidified. After solidification of media, we labeled each petri plate with permanent
marker.

Figure 4 Media pouring

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 Streaking:

After solidification, we performed the streaking process. For the streaking process, we
used an inoculation loop. We sterilized the inoculation loop in the spirit lamp by putting
the loop into flame until it became red hot. Then inserted the loop into solidified media in
Petri plates to become cool down a little bit. Now the spores on bread samples were
picked up with the help of the loop. Similarly we dipped the loop in homemade curd
sample and pick up microbes with the help of the loop. All this process occurred in the
presence of a turned-on condition of the spirit lamp inside the laminar hood.

Immediately streak the inoculating loop very gently at one side of Petri plate media and
form smear using back and forth motion. Again sterilize the loop using a spirit lamp and
allow it to cool by the same method, mentioned earlier. Going back to the edge that we
just streaked we performed streaking in a zigzag manner.

Figure 5 streaking

Incubation:

The plates were incubated in an upright position at 30˚C for 4-5days for fungal growth
and 37°𝐶 for bacterial growth.

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 Figure 6 Incubator

Microscopic examination:

A small portion of fungal colony was cut using a sterile scalpel. It was then place on a
sterile glass slide using a sterile forceps. A drop of lactophenol cotton blue stain was put
on slide containing fungi and covered with a cover slip. The excess stain was removed
and observed under the microscope.

Figure 7 Microscopic examination

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 Dual culture overlay assay method:

LAB were screened for antifungal activity using a dual-culture overlay assay. Bacteria
that were grow on nutrient agar plates were then overlaid with 10 ml of potato dextrose
agar containing 104 fungal spores per ml.

After 48 h of aerobic incubation at 30°C, the zone of inhibition was measured.

Figure 8 Agar overlay

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CHAPTER 4

Results:

This present study showed the isolation and microscopic examination of Rhizopus
stolonifer from spoiled bread made from different flours and its inhibition by lactic acid
bacteria isolated from homemade curd. 71 samples were collected .Fungal growth
occurred on plates after 24hr due to high temperature, about 40℃. Highest growth
occurred on barley samples. Similarly highest inhibition due to lactic acid bacteria
occurred in barley samples.

Table 1 Fungal growth on the basis of scale

Sample no. Barley Wheat Maize

1 3.5 mm 4 mm 4 mm

2 4 mm 4 mm 5 mm

3 4 mm 4 mm 3.5 mm

4 5.8 mm 4.5 mm 4 mm

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 Fungal growth on the basis of scale
7

Sample 1
4
Sample 2
3 Sample 3
Sample 4
2

0
Barley Wheat Maize

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 Culture examination:

White cottony mycelia, with black dots appeared on the fungal petri plate covered the
entire plate. After some days they become black producing spores in sporangium. Mucoid
whitish colonies of lactic acid bacteria appear on bacterial plates after 24 hrs.

Bacterial growth fungal growth Changed color of fungus

Figure 9 culture examination

Microscopic examination:

Under microscope sporangiospores were produced inside a spherical sporangium.

Columella was present on the top of the sporangiophore. Root-like rhizoids were found.
Following are different shapes of hyphae we seen on microscope.

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 Fungus on wheat

Fungus on barley

Fungus on maize

Figure 10 Microscopic examination

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 Fungus inhibition:

The inhibition was graded by relating the inhibited growth area per inoculation streak to
the total area of the petri dish. The inhibition area was also related to the variation in
length of the bacterial streak.

Figure 11 Fungus inhibition

Table 2 Rhizopus inhibition by LAB

Samples Diameter of inhibition zone (mm) ± SD

Wheat 1.5 mm±0.2

Maize 1.2 mm±0.4

Barley 1.5 mm±0.1

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 Fungal inhibition
2.5

1.5

Inhibition zone
1

0.5

0
Wheat Maize Barley

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CHAPTER
31
Discussion:
Nirmala and Pathmanathan (2017) collected twenty bread samples from different shops
in Jaffna. The fungi involved during spoilage were isolated and identified based on the
cultural and morphological characteristics. We also collected 71 samples of bread backed
from different flour brought from different shops in Bannu. We isolated and identified
Rhizopus stolonifer from spoiled bread based on the cultural and morphological
characteristics. Rhizopus stolonifer was cultured on Potato dextrose agar medium and
examined under microscope. Maximum growth was occurred on barley samples. Soma
Barman and Ranjan Ghosh (2017) collected one potent Mohammed Al-Haik and Ghadah
M. S isolates of LAB for antimicrobial activity on MRS medium revealed that the highest
antimicrobial activities were against Rhizopus stolonifer .Lactic acid bacterial strain C14
with strong antifungal activity was isolated from homemade curd. We also collected
lactic acid bacteria from homemade curd in order to inhibit fungal growth. LAB was
cultured on nutrient agar medium. Sengupta S, Mandal NC (2017) incubated plates at
28˚C for 48 h. Initially 125 colonies were taken randomly and were checked for their
antifungal potential against Rhizopus stolonifer by dual culture overlay method. Colonies
produced prominent zones of inhibition. We also incubated plates at 28˚C for 48 h and
observed fungal inhibition by LAB on petri plates by dual culture overlay method. We
measured zone of inhibition. Maximum inhibition was obvious in barley samples.

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CHAPTER
32
Conclusion:

The present study was aimed to observed inhibition of Rhizopus stolonifer by lactic acid
bacteria isolated from homemade curd. Rhizopus stolonifer was isolated from spoiled
bread samples. A total of 71 samples were cultured on potato dextrose agar for fungal
growth. Maximum fungal growth occurred on barley samples. LAB was cultured on
nutrient agar. By dual culture overlay method zone of inhibition was measured showing
fungal inhibition by LAB. Maximum inhibition was observed in barley samples. Thus,
LAB can be used as food biopreservative.

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 Future Prospective:

The future prospects of the present study will be to address other antifungal bacteria to be
used as biopreservative. Moreover, I will also work on the effects of antifungal bacteria
on the human being.

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 Recommendations:

The use of antifungal LAB instead of chemical preservatives would enable the food
industry to produce organic food without addition of chemical substances. In addition to
the already known excellent properties of LAB they could enhance the nutritional value
and prolong conservation of food. Thus addition of curd during baking process could
enhance shelf life of bread.

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