University of Khartoum Graduate Collage Medical & Health Studies

University of Khartoum

Graduate Collage

Medical & Health Studies Board

Fungal Contamination in Cheese at Khartoum North Markets

Hawyda Abdel Azim Abdel Alatif Mohammed

B.Sc. U.of Shendi (2004)

A thesis submitted in partial fulfillment for the requirements of the degree of master of Public and Environmental health (Food hygiene and safety)

Supervisor:

Dr/ Elrasheed A.Ali

B.Sc.Vet.M.Sc.Ph.D

Uof K

2010

` Didicailion

My Father and Mother

My beloved husband and Sondos

My brother and sisters

Acknowledgement

All thanks to my supervisor Dr.Alrasheed A.Ali for his supervision and valuable guidance.

I should also acknowledge with thanks the valuable help from the

Staff of the department of food Hygiene and safety

Table of content

Dedication...... I

Acknowledgment...... II

Table of contents...... III

Abbreviations...... VI

Abstract English...... VII

Abstract Arabic ...... IX

Chapter One

1.1. Introduction...... 1 1.1.1. Significance of study...... 1 1.1.2. Objective...... 2 1.2. Literature review...... 3 1.2.1. Etymology...... 4

1.2.2. History……………………………………………………………………………..4

1.2.3. Types...... 5

1.2.4. Fresh whey and stretched curd cheeses...... 6

1.2.5. Classified by texture...... 7

1.2.6. Classified by content...... 8

1.2.7. Soft ripened and blue vein...... 8

1.2.8. Processed cheeses...... 9

1.2.9. Eating and cooking...... 9

1.2.10. Health and nutrition...... 10

1.2.11. Mould...... 11

1.2.12. Biology...... 12

1.2.13. Categories of mould...... 13

1.2.14. Uses of mould...... 13

iii

1.2.15. Drug creation...... 14

1.2.16. Health effects...... 14

1.2.17. Toxic mould...... 15

1.2.18. Diseases transmitted by mould...... 17

1.2.19. Benefits of mould...... 19

1.2.20. Mould in processes...... 20

Chapter two

2.1. Materials and methods...... 21

2.1.1. Samples...... 21

2.1.1.2. Transport and keeping of collected samples...... 21

2.1.1.3.LaboratoryEquipment...... 21

2.1.3. Examination of mould...... 22

2.1.4. Examination of yeast...... 22

2.1.5. Identifiction of molud and yeast...... 22

Chapter three

3. Result...... 24

Chapter Four

4.1. Discussion...... 39

4.2. Conclusion...... 42

4.3. Recommendation...... 43

4.4. Reference...... 44

List of table Chapter three

Table3.1Minimum and maximum range of mould contamination in cheese compare with acceptable Sudanese Standers Metro logy Organization……………………………………………………………………………………..25

Table 3.2 Total viable counts of yeasts isolated from 100 samples of cheese collected from different retail sale in Khartoum North……………………………………………………………………………………...... 26

Table 3- 3Total viable count (T.V.C) of moulds isolated from 100 samples of cheese collected from different retail sale in Khartoum North………………………………………………………………………………………………29

List of figures

Chapter three

Figures (3.1) Slides culture of Aspergillus pink colony on sabouruad Dextrose Agar isolated

From 100 samples of cheese……………………………………………………………………………………32

Figures (3.2) Slides culture of Aspergillus fumigats green and dark colony on sabouruad Dextrose Agar isolated from 100 samples of cheese …………………………………………………………………………………32

Figures (3.3)Slides culture of saccharomyces white colony on sabouruad Dextrose Agar isolated from 100 samples of cheese ………………………………………………………………………………………………….33

Figures (3.4) Slides culture of Debaryomayces stained dark on sabouruad Dextrose Agar isolated from 100 samples of cheese ………………………………………………………………………………………………….33

Figures (3.5) Slides culture of Debaryomayces white colony on sabouruad Dextrose Agar isolated from 100 samples of cheese ……………………………………………………………………………………………………34

Figures (3.6) Slides culture of Aspergillus niger black colony on sabouruad Dextrose Agar isolated from 100 samples of cheese……………………………………………………………………………………………………34

Figures (3.7) Slides culture of Rhizopus dark pink on sabouruad Dextrose Agar isolated from 100 samples of cheese …………………………………………………………………………………………………………….35

Figures (3.8) Slides culture of Aspergillus fumigats green and dark colony on sabouruad Dextrose Agar isolated from 100 samples of cheese……………………………………………………………………………………..35

Figures (3.9) Slides culture of Debaryomayces stained dark on sabouruad Dextrose Agar isolated from 100 samples of cheese………………………………………………………………………………………………………36

Figures (3.10) Slides culture of saccharomyces white colony on sabouruad Dextrose Agar isolated from 100 samples of cheese ………………………………………………………………………………………………36

Abbreviations

pH Hydrogen ion concentration

USA United States of America

CFU|g Colony forming unit per gram

HIV: Hepatitis Immune Virus.

G.M.P: Good Manufacturing Bract ice.

HACCP: Hazard Analysis and Critical Control.

NACL: Sodium Chloride.

Abstract

Back ground: - Yeasts and moulds present a hazardous health problem in food, allowing outgrowth of both spoilage and pathogenic bacteria. Moulds are capable of forming toxins and causing mycotoxicoses in man and animals are distributed all over the world. They grow, produce toxins and multiply on a wide variety of foods over wide ranges of pH, temperature and humidity.

Design: - Descriptive, cross sectional study.

Study area:-Khartoum North locality – (Feb.2010 – Nov.2010).

Objective:- 1 – To study the fungal contamination of Sudanese cheese at Khartoum North. 2-To identify different species of fungi in cheese.

Methodology:

A hundred samples of 100 grams were collected randomly chosen shops from Khartoum locality and hygienically transferred to the Faculty of Public and Environment Health microbiology laboratory for the study. The laboratory sample is 25 gram should be between 100-500 gram.

1-Total viable count (T.V.C) for yeast and moulds were used to determine the hygienic level of cheese.

2- Identification of mould and yeast was carried using the phonotypical characteristics of moulds – (using standard F.A.O methods).

Result:

In the study the contamination percentages of cheese was as follows:

45% of all cheese was found contaminated by fungi. Mould species isolated from cheese samples were Aspergillus sp35%. Rhizopus sp10 %.

55% of cheese samples were contaminated by yeasts species, (Debaryomayces 35% and Saccharymyces

20 %).

vii

The total viable counts for microorganisms were:-

Moulds: 1x102---- 2x102 CFU/g

Yeasts : 1x102---- 9 x102 CFU/g

CONCLUSSIONS

When results were compared to the national (s.s.m.o) and international (Codex Alimentarius) The study concluded that: the cheese was highly contaminated and by different types of moulds and yeasts. This indicated that the cheese was produced under non hygienic conditions, storage or handling. Accordingly a number of recommendations from the Good Manufacturing Practice (G.M.P) and Hazard Analysis and Critical Control (HACCP)-as Stated by the Codex Alimentarius –were stated to improve the hygienic state of cheese in Khartoum North locality.

viii

ﺍﻟﻤﺴﺘﺨﻠﺹ

ﺍﻟﺨﻠﻔﻴﺔ:-

ﺍﻟﺨﻤﺎﺌﺭ ﻭﺍﻟﻔﻁﺭﻴﺎﺕ ﺘﻤﺜل ﻤﺨﺎﻁﺭ ﻭﻤﺸﺎﻜل ﺼﺤﻴﺔ ﻓﻰ ﺍﻷﻏﺫﻴﺔ . ﻭﻫﻰ ﺘﺴﻤﺢ ﺒﻨﻤﻭ ﺒﻌﺽ ﺍﻟﺒﻜﺘﺭﺒﺎ ﺍﻟﻤﻤﺭﻀﺔ ﻭﺍﻟﻤﺴﺒﺒﺔ ﻟﻠﻔﺴﺎﺩ. ﺍﻟﻔﻁﺭﻴﺎﺕ

ﻗﺎﺩﺭﺓ ﻋﻠﻰ ﺇﻨﺘﺎﺝ ﺍﻟﺴﻤﻭﻡ ﻤﺴﺒﺒﺔﻷﻤﺭﺍﺽ ﺍﻟﻤﺎﻴﻜﻭﺘﻭﻜﺴﻴﻥ ﻓﻲ ﺍﻹﻨﺴﺎﻥ ﻭﺍﻟﺤﻴﻭﺍﻥ ﻭﻫﻰ ﻤﻨﺘﺸﺭﺓ ﻓﻰ ﺠﻤﻴﻊ ﺃﻨﺤﺎﺀ ﺍﻟﻌﺎﻟﻡ. ﻭﻫﻰ ﺘﻨﻤﻭ ﻤﻨﺘﺠﺔ ﻟﻠﺴﻤﻭﻡ

ﻭﺘﺘﻀﺎﻋﻑ ﻓﻰ ﺍﻨﻭﺍﻉ ﻜﺜﻴﺭﻩ ﻤﻥ ﺍﻻﻏﺫﻴﺔ ﻭﻓﻰ ﻤﺩﻯ ﻜﺒﻴﺭ ﻤﻥ ﺩﺭﺠﺔ ﺍﻟﺤﻤﻭﻀﺔpH ﻭ ﺩﺭﺠﺎﺕ ﺍﻟﺤﺭﺍﺭﺓ ﻭﺍﻟﺭﻁﻭﺒﺔ.

ﺍﻟﺘﺼﻤﻴﻡ:- ﺩﺭﺍﺴﺔ ﻭﺼﻔﻴﺔ ﻤﻘﻁﻌﻴﺔ.

ﺍﻟﻤﻜﺎﻥ: - ﻤﺤﻠﻴﺔ ﺍﻟﺨﺭﻁﻭﻡ ﺒﺤﺭﻯ ﻓﺒﺭﺍﻴﺭ _ ﻨﻭﻓﻤﺒﺭ2010() .

ﺍﻻﻫﺩﺍﻑ:-

1- ﺩﺭﺍﺴﺔ ﺘﻠﻭﺙ ﺍﻻﺠﺒﺎﻥ ﺍﻟﺴﻭﺩﺍﻨﻴﺔ ﺒﺎﻟﻔﻁﺭﻴﺎﺕ (ﺍﻟﺨﺭﻁﻭﻡ ﺒﺤﺭﻯ).

2-ﺍﻟﺘﻌﺭﻑ ﻋﻠﻰ ﺍﻷﻨﻭﺍﻉ ﺍﻟﻤﺨﺘﻠﻔﺔ ﻟﻠﻔﻁﺭﻴﺎﺕ ﺍﻟﻤﻌﺯﻭﻟﺔ ﻤﻥ ﺍﻟﺠﺒﻨﻪ .

. ﻁﺭﻴﻘﺔ ﺍﻟﺒﺤﺙ:

* ﻤﺎﺌﺔ ﻋﻴﻨﺔ(100ﺠﺭﺍﻡ ﻟﻜل ) ﻤﻥ ﺍﻟﺠﺒﻨﺔ ﺠﻤﻌﺕ ﻤﻥ 100 ﻤﺭﻜﺯ ﺒﻴﻊ ﻤﻥ ﻤﺤﻠﻴﺔ ﺍﻟﺨﺭﻁﻭﻡ ﺒﺤﺭﻯ. ﺭﺤﻠﺕ ﺒﻁﺭﻴﻘﻪ ﺼﺤﻴﻪ ﺍﻟﻰ ﻤﻌﻤل ﺍﻷﺤﻴﺎﺀ

ﺍﻟﺩﻗﻴﻘﻪ ﺒﻜﻠﻴﺔ ﺍﻟﺼﺤﺔ ﺍﻟﻌﺎﻤﻪ ﻭﺼﺤﻪ ﺍﻟﺒﻴﺌﻪ ﻟﻠﺩﺭﺍﺴﻪ . ﺘﻡ ﺍﺨﺫ 25 ﺠﺭﺍﻡ ﻤﻥ ﻜل ﻋﻴﻨﻪ ﺒﻴﻥ ( 100 – 500 ﺠﺭﺍﻡ )

1 / ﺘﻡ ﺍﺴﺘﺨﺩﺍﻡ ﻁﺭﻴﻘﺔ ﺍﻟﻌﺩ ﺍﻟﻜﻠﻰ ﻟﻠﻔﻁﺭﻴﺎﺕ ﻭ ﺍﻟﺨﻤﺎﺌﺭ ﻟﺘﺤﺩﻴﺩ ﺍﻟﻤﺴﺘﻭﻯ ﺍﻟﺼﺤﻰ ﻟﻠﺠﺒﻨﺔ.

2/ ﺘﻡ ﺘﺤﺩﻴﺩ ﺃﻨﻭﺍﻉ ﺍﻟﻔﻁﺭﻴﺎﺕ ﻭﺍﻟﺨﻤﺎﺌﺭ ﻋﻥ ﻁﺭﻴﻕ ﺍﻟﻨﻤﻁ ﺍﻟﻅﺎﻫﺭﻯ ﻟﺨﺼﺎﺌﺹ ﺍﻟﻔﻁﺭﻴﺎﺕ - ( ﺍﺴﺘﺨﺩﺍﻡ ﻤﻌﺎﻴﻴﺭ ﻤﻨﻅﻤﺔ ﺍﻷﻏﺫﻴﺔ ﻭﺍﻟﺯﺭﺍﻋﺔ) .

ﺍﻟﻨﺘﺎﺌﺞ:-

* ﻓﻰ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻨﺴﺏ ﺘﻠﻭﺙ ﺍﻻﺠﺒﺎﻥ ﻜﺎﻻﺘﻰ:-

* 45.|. ﻤﻥ ﺍﻻﺠﺒﺎﻥ ﻤﻠﻭﺜﻪ ﺒﺎﻟﻔﻁﺭﻴﺎﺕ ( ﻋﻭﺍﺌل ﺍﻟﻔﻁﺭﻴﺎﺕ ﺍﺴﺒﻴﺭﻭﺠﻴﻠﺱ35.|. ﻭ ﺭﺍﻴﺯﻭﺒﻴﺱ10.|.) .

* 55 .|. ﻤﻥ ﺍﻻﺠﺒﺎﻥ ﻤﻠﻭﺜﺔ ﺒﺎﻟﺨﻤﺎﺌﺭ( ﻋﻭﺍﺌل ﺍﻟﺨﻤﺎﺌﺭ Debaryomayce 35.|. ﻭ 20Saccharymyces.|.) .

* ﺍﻟﻌﺩ ﺍﻟﻜﻠﻰ ﺍﻟﺤﻰ ﻟﻠﺘﻠﻭﺙ ﺍﻟﻤﻴﻜﺭﻭﺒﻰ ﻜﺎﻻﺘﻰ:-

ﻓﻄﺮﻳﺎت: - x2 ---210x1 210 ﺨﻠﻴﻪ / ﺠﻡ

ﺨﻤﺎﺌﺭ: - 210x9 ---210 x 1 ﺨﻠﻴﻪ / ﺠﻡ

ﺍﻟﺨﻼﺼﺔ:

ﻋﻨﺩ ﻤﻘﺎﺭﻨﻪ ﺍﻟﻨﺘﺎﺌﺞ ﺒﺎﻟﻤﻭﺍﺼﻔﺎﺕ ﺍﻟﻌﺎﻟﻤﻴﻪ ﺍﻟﺼﺎﺩﺭﻩ ﻋﻥ ﻤﻭﺍﺼﻔﺎﺕ ﻤﻨﻅﻤﻪ ﺍﻻﻏﺫﻴﻪ ﺍﻟﻌﺎﻟﻤﻴﻪ , ﺨﻠﺼﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺍﻟﻰ ﺍﻥ ﺍﻻﺠﺒﺎﻥ ﻋﺎﻤﺔ ﻭﻓﻰ ﻤﺤﻠﻴﺔ

ﺍﻟﺨﺭﻁﻭﻡ ﺒﺤﺭﻯ ﺨﺎﺼﻪ ﻤﻠﻭﺜﺔ ﺒﺩﺭﺠﻪ ﻋﺎﻟﻴﻪ ﺒﺄﻨﻭﺍﻉ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﺍﻟﻔﻁﺭﻴﺎﺕ ﻭﺍﻟﺨﻤﺎﺌﺭ ﺍﻟﺘﻰ ﻗﺩ ﺘﻜﻭﻥ ﺨﻁﺭﺓ ﻋﻠﻰ ﺼﺤﺔ ﺍﻻﻨﺴﺎﻥ ﻭﻫﺫﺍ ﻴﻌﺘﺒﺭ

ﻤﺅﺸﺭ ﺍﻟﻰ ﺍﻥ ﺍﻟﺠﺒﻨﺔ ﺘﻡ ﺍﻨﺘﺎﺠﻬﺎ ﺍﻭ ﺘﺨﺯﻴﻨﻬﺎ ﺍﻭﺘﺩﺍﻭﻟﻬﺎ ﺒﻁﺭﻴﻘﺔ ﻏﻴﺭ ﺼﺤﻴﺔ. ﻭﻭﻓﻘﺎ ﻟﺫﻟﻙ ﺘﻡ ﻭﻀﻊ ﻋﺩﺩ ﻤﻥ ﺍﻟﺘﻭﺼﻴﺎﺕ ﻁﺒﻘﺎ ﻟﻠﻤﻤﺎﺭﺴﺎﺕ ﺍﻟﺘﺼﻨﻴﻌﻴﺔ

ﺍﻟﺠﻴﺩﺓ ﻭﺘﺤﺩﻴﺩ ﻨﻘﺎﻁ ﺍﻟﺘﺤﻜﻡ ﺍﻟﺤﺭﺠﺔ ﻭﺍﻟﺘﻰ ﻭﻀﻌﺘﻬﺎ ﻤﻨﻅﻤﺔ ﺍﻻﻏﺫﻴﺔ ﻟﺘﺤﺴﻴﻥ ﺍﻟﺤﺎﻟﺔ ﺍﻟﺼﺤﻴﺔ ﻻﻨﺘﺎﺝ ﺍﻟﺠﺒﻨﺔ.

1.1. INTRODUCTION

Cheese can be defined as a consolidated curd of milk solids in which milk fat is entrapped by coagulated casein. Unlike fermented milks, the physical characteristics of cheese are far removed from those of milk. (James. et al. 2005)

This is because protein coagulation proceeds to a greater extent as a result of the use of photolytic enzymes and much of the water content of the milk separates and is removed in the form of whey. Typically the yield of cheese from milk is of the order of 10%. (James. et al. 2005)

In general, cheese supplies a great deal of calcium, protein, phosphorus and fat. A 30- gram (1.1 oz) serving of Cheddar cheese contains about 7 grams (0.25 oz) of protein and 200 milligrams of calcium. Nutritionally, cheese is essentially concentrated milk: it takes about 200 grams (7.1 oz) of milk to provide that much protein, and 150 grams (5.3 oz) to equal the calcium. Some studies claim that cheddar, mozzarella, Swiss and American cheeses can help to prevent tooth decay. The calcium, protein, and phosphorus in cheese may act to protect tooth enamel. • Cheese increases saliva flow, washing away acids and sugars. • Cheese may have an antibacterial effect in the mouth. (Mellgren, Jame, 2003). 1.1.1. Significance of study:- The yeasts and moulds are most likely to be found in the foods in which the environment is less favorable for bacteria growth, e.g. low pH, low moisture, high salt or sugar content, low storage temperature, the presence of antibiotics, or exposure to irradiation. Yeasts and moulds present a problem in foods in that they discolor food surfaces, cause off-odors and off-flavors, because various degrees of spoilage alter substrates allowing for the outgrowth of pathogenic bacteria, and are able to produce mycotoxins in certain instances.

1.1.2. Objective

General objective:-

Types of Fungi Encountered in Cheese at Khartoum North markets.

Specific objective:-

1 – To study the fungal contamination of Sudanese cheese at Khartoum North. 2-To identify the different species of fungi isolated from cheese.

1.2. Literature review

Cheese is a generic term for a diverse group of milk-based food products. Cheese is produced throughout the world in wide-ranging flavors, textures, and forms.

Cheese consists of proteins and fat from milk, usually the milk of cows, buffalo, goats, or sheep. It is produced by coagulation of the milk protein casein. Typically, the milk is acidified and addition of the enzyme rennet causes coagulation. The solids are separated and pressed into final form. Some cheeses have moulds on the rind or throughout. Most cheeses melt at cooking temperature.

Hundreds of types of cheese are produced. Their styles, textures and flavors depend on the origin of the milk (including the animal’s diet), whether they have been pasteurized, the butterfat content, the bacteria and mould, the processing, and aging. Herbs, spices, or wood smoke may be used as flavoring agents. The yellow to red color of many cheeses is from adding annatto.

For a few cheeses, the milk is curdled by adding acids such as vinegar or lemon juice. Most cheeses are acidified to a lesser degree by bacteria, which turn milk sugars into lactic acid, and then the addition of rennet completes the curdling. Vegetarian alternatives to rennet are available; most are produced by fermentation of the fungus Mucor miehei, but others have been extracted from various species of the Cynara thistle family.

Cheese is valued for its portability, long life, and high content of fat, protein, calcium, and phosphorus. Cheese is more compact and has a longer shelf life than milk. Cheese makers near a dairy region may benefit from fresher, lower-priced milk, and lower shipping costs. The long storage life of some cheese, especially if it is encased in a protective rind, allows selling when markets are favorable, (Fankhauser, David, 2007)

1.2.1. Etymology

The word cheese ultimately comes from Latin case us, from which the modern word casein is closely derived. The earliest source is from the proto-Indo-European root kwat, which means "to ferment, become sour".

More recently, cheese comes from chese in English and cīese or cēse in Old English. Similar words are shared by other West Germanic languagesWest Frisiantsiis, Dutchkaas, GermanKäse, Old High Germanchāsi all from the reconstructed West-Germanic form kasjus, which in turn is an early borrowing from Latin.

When the Romans began to make hard cheeses for their legionaries' supplies, a new word started to be used: formaticum, from case us formatusor "moulded cheese" (as in "formed", not "mouldy"). It is from this word that we get the French fromage, Italian formaggio, Catalan formatge, Breton fourmaj and Provencal furmo. Cheese itself is occasionally employed in a sense that means "moulded" or "formed". Head cheese uses the word in this sense (Simpson, 1979).

1.2.2. History:-

Origins

Cheese is an ancient food whose origins predate recorded history. There is no conclusive evidence indicating where cheese making originated, either in Europe, Central Asia or the Middle East, but the practice had spread within Europe prior to Roman times and, according to Pliny the Elder, had become a sophisticated enterprise by the time the Roman Empire came into being ( Ensrud, Barbara .1981)

Proposed dates for the origin of cheese making range from around 8000 BCE (when sheep were first domesticated) to around 3000 BCE. The first cheese may have been made by people in the Middle East or by nomadic tribes in Central Asia. Since animal skins and inflated internal organs have, since ancient times, provided storage vessels for a

range of foodstuffs, it is probable that the process of cheese making was discovered accidentally by storing milk in a container made from the stomach of an animal, resulting in the milk being turned to curd and whey by the rennet from the stomach. There is a legend with variations about the discovery of cheese by an Arab trader who used this method of storing milk.

Cheese making may have begun independently of this by the pressing and salting of curdled milk in order to preserve it. Observation that the effect of making milk in an animal stomach gave more solid and better-textured curds, may have led to the deliberate addition of rennet.

The earliest archeological evidence of cheese making has been found in Egyptian tomb murals, dating to about 2000 BCE. The earliest cheeses were likely to have been quite sour and salty, similar in texture to rustic cottage cheese or feta, a crumbly, flavorful Greek cheese.

Cheese produced in Europe, where climates are cooler than the Middle East, required less salt for preservation. With less salt and acidity, the cheese became a suitable environment for useful microbes and moulds, giving aged cheese their pronounced and interesting flavors (Ensrud, Barbara .1981)

1.2.3. Types:-

Factors in categorization

Factors which are relevant to the categorization of cheeses include:

• Length of aging • Texture • Methods of making • Fat content • Kind of milk • Country/Region of Origin

List of common categories

• fresh • whey • pasta filata • semi-soft • semi-firm • hard • double and triple cream • soft-ripened • blue vein • goat or sheep • sharp • processed

1.2.4. Fresh, whey and stretched curd cheeses:-

The main factor in the categorization of these cheeses is their age. Fresh cheeses without additional preservatives can spoil in a matter of days.

For these simplest cheeses, milk is curdled and drained, with little other processing. Examples include cottage cheese, Romanian Cash, Neufchatel and fresh goat's milk chèvre. Such cheeses are soft and spreadable, with a mild taste.

Whey cheeses are fresh cheeses made from the whey discarded while producing other cheeses. Provencal Brousse, Corsican Brocciu, Italian Ricotta, RomanianUrda, Greek Mizithra, and NorwegianGeitost are examples. Brocciu is mostly eaten fresh, and is as such a major ingredient in Corsican cuisine, but it can be aged too.

Traditional pasta filata cheeses such as Mozzarella also fall into the fresh cheese category. Fresh curds are stretched and kneaded in hot water to form a ball of Mozzarella, which in southern Italy is usually eaten within a few hours of being made. Stored in

brine, it can be shipped, and is known worldwide for its use on pizzas. Other firm fresh cheeses include paneer and queso fresco.

1.2.5. Classified by texture:-

Categorizing cheeses by firmness is a common but inexact practice. The lines between soft, semi-soft, semi-hard, and hard are arbitrary, and many types of cheese are made in softer or firmer variations. The factor controlling the hardness of a cheese is its moisture content which is dependent on the pressure with which it is packed into moulds and the length of time it is aged.

Semi-soft cheeses and the sub-group, Monastery cheeses have high moisture content and tend to be bland in flavor. Some well-known varieties include Havarti, Munster and Port Saluted.

Cheeses that range in texture from semi-soft to firm include Swiss-style cheeses like Emmental and Gruyère. The same bacteria that give such cheeses their holes also contribute to their aromatic and sharp flavors. Other semi-soft to firm cheeses includes Gouda, Edam, Jarlsberg and Cantal. Cheeses of this type are ideal for melting and are used on toast for quick snacks (Jenkins, Steven, 1996).

Harder cheeses have lower moisture content than softer cheeses. They are generally packed into moulds under more pressure and aged for a longer time. Cheeses that are semi-hard to hard include the familiar Cheddar, originating in the village of Cheddar in England but now used as a generic term for this style of cheese, of which varieties are imitated worldwide and are marketed by strength or the length of time they have been aged. Cheddar is one of a family of semi-hard or hard cheeses (including Cheshire and Gloucester) whose curd is cut, gently heated, piled, and stirred before being pressed into forms. Colby and Monterey Jack are similar but milder cheeses; their curd is rinsed before it is pressed, washing away some acidity and calcium. A similar curd-washing takes place when making the Dutch cheeses Edam and Gouda.

Hard cheeses “grating cheeses" such as Parmesan and Romano are quite firmly packed into large forms and aged for months or years. (Jenkins, Steven, 1996).

1.2.6. Classified by content:-

Some cheeses are categorized by the source of the milk used to produce them or by the added fat content of the milk from which they are produced. While most of the world's commercially available cheese is made from cows' milk, many parts of the world also produce cheese from goats and sheep, well-known examples being Roquefort, produced in France, and Pecorino Romano, produced in Italy, from ewe's milk. One farm in Sweden also produces cheese from moose's milk. Sometimes cheeses of a similar style may be available made from milk of different sources Feta style cheeses, for example, are made from goats' milk in Greece and from sheep and cows' milk elsewhere.

Double cream cheeses are soft cheeses of cows' milk which are enriched with cream so that their fat content is 60% or, in the case of triple creams, 75% (Jenkins, Steven, 1996).

1.2.7. Soft ripened and blue vein:-

There are three main categories of cheese in which the presence of mould is a significant feature: soft ripened cheeses, washed rind cheeses and blue cheese.

Soft-ripened cheeses are those which begin firm and rather chalky in texture but are aged from the exterior inwards by exposing them to mould. The mold may be a velvety bloom of Penicillium candida or P. camemberti that forms a flexible white crust and contributes to the smooth, runny, or gooey textures and more intense flavors of these aged cheeses. Brie and Camembert, the most famous of these cheeses, are made by allowing white mould to grow on the outside of a soft cheese for a few days or weeks. Goats' milk cheeses are often treated in a similar manner, sometimes with white moulds (Chèvre- Boîte) and sometimes with blue.

Washed-rind cheeses are soft in character and ripen inwards like those with white mould

However, they are treated differently. Washed rind cheeses are periodically cured in a solution of saltwater brine and other mould-bearing agents which may include beer, wine, brandy and spices, making their surfaces amenable to a class of bacteria Brevibacterium linens which impart pungent odors and distinctive flavors. Washed-rind cheeses can be soft (Limburger), semi-hard (Munster), or hard (Appenzeller). The same bacteria can also have some impact on cheeses that are simply ripened in humid conditions, like Camembert.

So-called blue cheese is created by inoculating a cheese with Penicillium roqueforti or Penicillium glaucum. This is done while the cheese is still in the form of loosely pressed curds, and may be further enhanced by piercing a ripening block of cheese with skewers in an atmosphere in which the mould is prevalent. The mould grows within the cheese as it ages. These cheeses have distinct blue veins which gives them their name, and, often, assertive flavors. The moulds may range from pale green to dark blue, and may be accompanied by white and crusty brown moulds. Their texture can be soft or firm. Some of the most renowned cheeses are of this type, each with its own distinctive color, flavor, texture and smell. They include Roquefort, Gorgonzola, and Stilton. (McGee, Harold, 2004).

1.2.8. Processed cheeses:-

Processed cheese is made from traditional cheese and emulsifying salts, often with the addition of milk, more salt, preservatives, and food coloring. It is inexpensive, consistent, and melts smoothly. It is sold packaged and either pre-sliced or unsliced, in a number of varieties. It is also available in aerosol cans. (McGee, Harold, 2004).

1.2.9. Eating and cooking:-

At refrigerator temperatures, the fat in a piece of cheese is as hard as unsoftened butter, and its protein structure is stiff as well. Flavor and odor compounds are less easily liberated when cold. For improvements in flavor and texture, it is widely advised that

cheeses be allowed to warm up to room temperature before eating. If the cheese is further warmed, to 26–32 °C (79–90 °F), the fats will begin to "sweat out" as they go beyond soft to fully liquid.

Above room temperatures, most hard cheeses melt. Rennet-curdled cheeses have a gel- like protein matrix that is broken down by heat. When enough protein bonds are broken, the cheese itself turns from a solid to a viscous liquid. Soft, high-moisture cheeses will melt at around 55 °C (131 °F), while hard, low-moisture cheeses such as Parmesan remain solid until they reach about 82 °C (180 °F). Acid-set cheeses, including halloumi, paneer, some whey cheeses and many varieties of fresh goat cheese, have a protein structure that remains intact at high temperatures. When cooked, these cheeses just get firmer as water evaporates.

Some cheeses, like raclette, melt smoothly; many tend to become stringy or suffer from a separation of their fats. Many of these can be coaxed into melting smoothly in the presence of acids or starch. Fondue, with wine providing the acidity, is a good example of a smoothly melted cheese dish. (McGee, Harold, 2004).

1.2.10. Health and nutrition:-

Some studies claim that cheddar, mozzarella, Swiss and American cheeses can help to prevent tooth decay. The calcium, protein, and phosphorus in cheese may act to protect tooth enamel.

• Cheese increases saliva flow, washing away acids and sugars. • Cheese may have an antibacterial effect in the mouth. (Mellgren, etal, 2003)

Mould

1.2.11 .Mould

Moulds are filamentous fungi that grow in the form of a tangled mass that spreads rapidly and may cover several inches of area in 2 to 3 days. The total of the mass or any large portion of it is referred to as mycelium. Mycelium is composed of branches or filaments referred to as hyphae. Those of greatest importance in foods multiply by ascospores, zygospores, or conidia. The ascospores of some genera are notable for their extreme degrees of heat resistance. One group forms pycnidia or acervuli (small, flask- shaped, fruiting bodies lined with conidiophores). Arthrospores result from the fragmentation of hyphae in some groups.

There were no radical changes in the systematics of food borne fungi during the 1980s. The most notable changes involve the discovery of the sexual or perfect states of some well-known genera and species. In this regard, the ascomycete state is believed by mycologists to be the more important reproductive state of a fungus, and this state is referred to as the teleomorph. The species name given to a teleomorph takes precedence over that for the anamorph, the imperfect or conidial state. Holomorph indicates that both states are known, but the teleomorph name is used. (Jay, 2003)

Mould includes species of microscopic fungi that grow in the form of multicellular filaments, called hyphae. In contrast, microscopic fungi that grow as single cells are called yeasts. A connected network of these tubular branching hyphae has multiple, genetically identical nuclei and is considered a single organism, referred to as a colony or in more technical terms a mycelium.

Moulds do not form a specific taxonomic or phylogenetic grouping, but can be found in the divisions Zygomycota, Deuteromycota and Ascomycota. Although some moulds cause disease or food spoilage, others are useful for their role in biodegradation or in the production of various foods, beverages, antibiotics and enzymes . (Madigan, Martinko, 2005)

1.2.12. Biology:-

There are thousands of known species of moulds, which include opportunistic pathogens, saprotrophs, aquatic species, and thermophiles. Like all fungi, moulds derive energy not through photosynthesis but from the organic matter in which they live. Typically, moulds secrete hydrolytic enzymes, mainly from the hyphal tips. These enzymes degrade complex biopolymers such as starch, cellulose and lignin into simpler substances which can be absorbed by the hyphae. In this way, moulds play a major role in causing decomposition of organic material, enabling the recycling of nutrients throughout ecosystems. Many moulds also secrete mycotoxins which, together with hydrolytic enzymes, inhibit the growth of competing microorganisms.

Moulds reproduce through small spores, which may contain a single nucleus or be multinucleate. Mould spores can be asexual (the products of mitosis) or sexual (the products of meiosis); many species can produce both types. Some can remain airborne indefinitely, and many are able to survive extremes of temperature and pressure.

Although moulds grow on dead organic matter everywhere in nature, their presence is only visible to the unaided eye when mould colonies grow. A mould colony does not comprise discrete organisms, but an interconnected network of hyphae called a mycelium. Nutrients and in some cases organelles may be transported throughout the mycelium. In artificial environments like buildings, humidity and temperature are often stable enough to foster the growth of mould colonies, commonly seen as a downy or furry coating growing on food or other surfaces.

Many moulds can begin growing at 4 °C (39 °F), the temperature within a typical refrigerator, or less. When conditions do not enable growth, moulds may remain alive in a dormant state depending on the species, within a large range of temperatures before they die. The many different mould species vary enormously in their tolerance to temperature and humidity extremes. Certain moulds can survive harsh conditions such as the snow- covered soils of Antarctica, refrigeration, highly acidic solvents, and even petroleum products such as jet fuel. (Roostita, 1996).

1.2.13. Categories of moulds:-

Two categories of organisms are presented here, the first being some miscellaneous genera that are found in some foods but are generally not regarded as significant. These are Cephalosporium, Diplodia, and Neurospora. Cephalosporium is a deuteromycete often found on frozen foods.

The microspores of some Fusarium species are similar to those of this genus. Diplodia is another deuteromycete that causes stem-end rot of citrus fruits and water tan-rot of peaches. Neurospora is an ascomycete and N. intermedia are referred to as the “red bread” mould. Monilia sitophila is the anamorph of N.intermedia. The latter is important in the oncom fermentation and has been found on meats. The “white spot” of beef is produced by Sporotrichum spp.; and rots of various fruits are caused by Gloeosporium spp. Some Helminthosporium spp. is plant pathogens and some are saprophytes.

The second category consists of xerophilic moulds, which are very important as spoilage organisms. In addition to Aspergillus and Eurotium, Pitt and Hocking37 include six other genera among the xerophiles:

Basipetospora, Chrysosporium, Eremascus, Polypaecilum, Wallemia, and Xeromyces. (Adams,etal, 2008).

1.2.14. Uses of mould:-

Food production Cultured moulds are used in the production of foods, including:

• Cheese (Penicillium spp.) • tempeh (Rhizopus oligosporus) • oncom (Neurospora sitophila) • Quorn (Fusarium venenatum) • bread • sausages

• soy sauce

The koji moulds are a group of Aspergillus species, notably Aspergillus oryzae, that have been cultured in eastern Asia for many centuries. They are used to ferment a soybean and wheat mixture to make soybean paste and soy sauce. They are also used to break down the starch in rice (saccharification) in the production of sake and other distilled spirits. (Stahnke, Sunesen, 2003)

Red rice yeast is a product of the mould Monascus purpureus grown on rice, and is common in Asian diets. The yeast contains several compounds collectively known as monoclines, which are known to inhibit cholesterol synthesis (David, 1968)

1.2.15. Drug creation: Alexander Fleming's famous discovery of the antibiotic penicillin involved the mould Penicillium chrysogenum.

Several cholesterol-lowering drugs (such as Lovastatin, from Aspergillus terreus) are derived from moulds.

The immunosuppressant drug cyclosporine, used to suppress the rejection of transplanted organs, is derived from the mould. . (National Institute for Occupational Safety and Health, 2008)

1.2.16. Health effects:- Moulds are ubiquitous in nature, and mould spores are a common component of household and workplace dust. However, when mould spores are present in large quantities, they can present a health hazard to humans, potentially causing allergic reactions and respiratory problems.

Some moulds also produce mycotoxins that can pose serious health risks to humans and animals. Some studies claim that exposure to high levels of mycotoxins can lead to neurological problems and in some cases death. Prolonged exposure,

E.g. daily workplace exposure may be particularly harmful. The term toxic mould refers to moulds that produce mycotoxins, such as Stachybotrys chartarum, and not to all

moulds in general. Mould in the home can usually be found in damp, dark or steam filled areas e.g. bathroom or kitchen, over cramped storage areas, recently flooded areas, basement areas, plumbing spaces, areas with poor ventilation and outdoors in humid environments. Symptoms caused by mould allergy are watery, itchy eyes, a chronic cough, headaches or migraines, difficulty breathing, rashes, tiredness, sinus problems, nasal blockage and frequent sneezing. In extremely rare cases, over-exposure to mould may result in bucal mould growth leading to death by asphyxiation. .

In the indoor environment, moulds are an unwelcome visitor. They can cause structural damage by decomposing wood, drywall, carpeting and other organic building materials, and can also cause health problems. Moulds generally cause health symptoms by producing allergens, toxic substances (mycotoxins), or by causing infections. Allergic responses to mould include hay fever-type symptoms, such as sneezing, runny nose, respiratory irritation, eye irritation, and skin rash. Mould can also cause asthma attacks. Moulds can cause infections by actively growing in the body. Moulds of the genus Aspergillus, for instance, can cause a condition known as aspergillosis, by growing in the lungs. This infection is almost always seen in individuals with a compromised immune system. Finally, certain moulds can produce toxins. These toxins have been reported to cause such symptoms as nose bleeding, lung bleeding, nausea, vomiting, and neurological disorders, such as memory loss. However, these symptoms remain unproven in respect to inhalation. (National Institute for Occupational Safety and Health, 2008)

1.2.17. Toxic mould:-

The fungi are heterotrophic and feed by absorption of soluble nutrients and although many fungi can metabolize complex insoluble materials, such as lignocelluloses, these materials have to be degraded by these creation of appropriate enzymes outside the wall. A number of fungi are parasitic on both animals, plants and other fungi, and some of these 280 Non-bacterial Agents of Food borne Illness parasitic associations have become very complex and even obligate.

However, it is the ability of some moulds to produce toxic metabolites, known as mycotoxins, in foods and their association with a range of human diseases, from gastro enteric conditions to cancer, which concerns us here.

The filamentous fungi grow over and through their substrate by processes of hyphen tip extension, branching and anastomosis leading to the production of an extensive mycelium. Some species have been especially successful in growing at relatively low water activities which allows them to colonize commodities, such as cereals, which should otherwise be too dry for the growth of micro-organisms.

Frequently, when moulds attack foods they do not cause the kind of putrefactive breakdown associated with some bacteria and the foods may be eaten despite being mouldy and perhaps contaminated with mycotoxins. Indeed, some of the changes brought about by the growth of certain fungi on a food may be organoleptically desirable leading to the manufacture of products such as mould-ripened cheeses and mould ripened sausages using species of Penicillium. (James, etal, 2005)

Mycotoxins are chemicals that are sometimes produced by various species of toxic mould. These toxins are real and are powerful weapons used by toxic moulds in a sort of microbial warfare to help them compete against bacteria and other moulds. Various moulds including but not limited to toxic black mould also known as Stachybotrys or toxic mould are common in Florida, but mould spores in residential settings, even if they are potentially toxic producing types and at high levels, are not automatically at high enough levels to result in toxic effects on human’s inhalation. It takes a lot of inhaled spores to poison a person. Currently, disagreement exists as to if residential mould spore exposures levels are ever high enough to result in toxic effect on humans. Toxic effects of mould mycotoxins in humans and farm animals leading to serious illness and even death via accidental ingestion of toxic mould .Effects of heavy exposure to mould toxins are many, but just two of the more common effects are immune suppression and liver cancer. Mycotoxins are believed to result in headaches, sore throats, hair loss, flu symptoms, diarrhea, fatigue, dermatitis, general malaise (tiredness) and psychological depression. (Davis, 1976)

Mycotoxins and Mycophagy:

The vegetative structures of the filamentous fungi are essentially based on the growth form of the spreading, branching, anastomosing mycelium and have a relatively limited morphological diversity. (James, et al, 2005)

1.2.18. Diseases transmitted by mould:-

Although moulds are a natural part of our environment, exposure to certain types of airborne mould spores can cause allergic reactions, asthma episodes and other respiratory problems. In addition, exposure to high spore levels can cause the development of an allergy to the mould. Health impacts from mold/mildew occur when individuals are exposed to large doses of mycotoxins, which are by-products produced from the moulds. Adverse health effects from exposure to large doses of mycotoxins, which are by- products produced from the moulds. Adverse health effects from exposure to mycotoxins may differ from person to person. While some people may be highly sensitive to mycotoxins and experience adverse health effects, other people exposed to the same dosage of the mould will be unaffected. The mere presence of mould found indoors does not indicate a public health risk.

Certain types of mould are considered toxic and can cause serious health effects. These species include Stachybotrys chartarum, Aspergillus versicolor, A. flavus,

A. fumigatus and Fusarium moniliforme. Stachybotyrs Chartarum is a greenish black mould that forms in environments with high moisture such as those that have water damage, water leaks, excessive humidity, condensation and flooding. The health effects of Stachybotyrs Chartarum range from short-term irritation to extremely serious illnesses. Stachybotrys chartarum can produce mycotoxins that can develop into an indoor air quality problem. Stachybotrys chartarum has the potential to cause symptoms such as coughing, wheezing, runny nose, irritated eyes or throat, skin rash or diarrhea.

Certain population groups have a greater health risk when exposed to mould. These people include infants and children, elderly, immune compromised patients, pregnant 17

women and individuals with respiratory conditions. Pathogenic mould can cause serious health effects in persons with suppressed immune systems, those taking chemotherapy, or those with HIV/AIDS. Allergenic moulds, normally not dangerous, can cause allergy or asthma symptoms (James, et al, 2005).

Allergy asthma and hypersensitivity diseases:-

Allergic reaction occurs when your body's immune system mistakes harmless proteins in mould spores or other allergens as if these proteins were harmful microbes trying to infect your body. Your body's immune system, feeling threatened releases histamines into the blood stream and these histamines are what actually causes coughing, sneeze, and watering of the eyes. Other person's immune systems will not mistake proteins in mould spores as a microbial threat, and thus not develop allergic reactions. Asthma is a condition where the small air sack like structures in the lungs called alveoli can contract and exhale air, but cannot properly expand to bring in new air. Moulds as well as other substances are common triggers of asthma. Mould-related allergenic and asthmatic conditions in homes are very common and very serious and should be addressed and not ignored. Many serious hypersensitivity diseases in humans, such as baker's lung, wood workers lung and others are the result of exposures to moulds by persons working in industries that result in long term exposure to elevated spore levels. hypersensitivity disease that results from the long term repeated exposure to elevated mould spore levels or other antigens, this condition can occur at in industry or agriculture where spore levels are high, it may also occur in mouldy offices or homes, it produces pneumonia like symptoms with fever, cough, tightness of chest, lung \infiltrates, and difficulty breathing. Once sensitized, individuals may react to extremely low, often un measurable, concentrations of antigenic materials.

Organic dust toxic syndrome is flu like illness that results from a short term exposure to very high levels of spores, such as the levels that may be encountered by workers doing large mould remediation jobs without wearing protective respirators. Symptoms show up several hours or a day or so after exposure and symptoms go away after a day or a few days. Building related illness is a term used to describe an illness such as one of the

above that results from time spent in a building around moulds or other allergens, irritants, or toxins, and sick building syndrome is used to describe a building related illness whose specific cause remains a mystery. (Davis, 1976)

Chronic Disease Caused by Mould: Mycoses

Mycosis is the general term for chronic disease caused by species of mould capable of inhabiting the human body or specific organs of and therefore causing disease. Mycosis are of long duration and very difficult to treat. The most often affected are airways, lungs, eyes, digestive tract, and skin. Ultimately they can spread throughout the body, especially when the immune system is weak.

Occupational medicine describes a large number of mycosis affecting people who work in environments with mould such as: Alter aria, wood-pulp worker's lung. Aspergillum, malt worker's lung. Penicillium, asthma of the cheese worker, and many others. These diseases are not necessarily specific to occupations and anyone can be affected. Children growing up in houses with mouldy basements tend to develop a large array of health problems and unfortunately many will suffer permanent health damage.

Volatile Organic Compounds:-

Mould cells release in the air volatile organic compounds. These are responsible for the musty odor present in mould infested houses or spaces. Volatile organic compounds are not mycotoxines, but they can cause symptoms of intoxication such as headaches, nausea, vomiting, and fatigue. The symptoms vanish after the person leaves the mould infested environment and are not known to leave consequences. (Haas, 2009)

• 1.2.19. Benefits of Mould:- Mould – A Natural Recycling Process

Mould can be destructive, but they are also beneficial. They help to make cheese, fertilize gardens, and speed decaying of garbage and fallen leaves. Today thousands of different types of mould fungi grow on and absorb food from substances such as soil, wood, decaying organic

matter, or living plants and other organisms. After the devastating forest fires in Yellowstone National Park, mould was the first on the scene to begin the nature’s process of decaying the leftover matter and building a base for new plants and trees.

1.2.20. Mould in Processes

Certain types of mould have proven extremely valuable in the synthesis of antibiotics and hormones used in medicine and of enzymes used in certain manufacturing processes. Penicillin, a product of the green mould P. notatum, revolutionized antibiotic drugs after its discovery in 1929, and the red bread mould Neurospora is an important tool in genetic experiments.

An interesting fact is if a medicine name ends in "mycin”, you know it was made with mould. Another is that in a few certain kinds of cheeses, mould is added for flavor. Butchers also inject mould into animals before they butcher to preserve the animal. Without mould, we would not have beer or bread. Molds also have many industrial uses, such as in the fermentation of organic acids and cheeses. Some fungi, such as mushrooms and truffles, are considered tasty delicacies that enhance a wide variety of recipes, including pizza. Other moulds can damage agricultural crops, cause disease in animals and humans, and form poisonous toxins in food. (David, 1968)

MATERIALS AND METHODS

• 2.1. 1. Samples:

A total of 100 samples of cheeses were collected from different retail sales in Khartoum North in plastic bags.

• 2.1.1.2. Transport and keeping of collected samples:

According to Harrigan and McCance (1976) samples were collected in ice box and transferred to the laboratory as fast as possible and kept refrigerated at about 4ºc until tested.

• 2.1.1.3. Laboratory Equipment Used:

The laboratory equipments include sensitive balance, Petri dishes or plates (90 x 20 mm) , pipettes ,test tubes, flask (250 ml), cover glass, slide, foil , flam ,cotton, tips, loop, (KOH 2%), antibiotic.

• 2.1.2.laboratory testes:-

• Preparation of dilutions: - a laboratory was conducted on the (100) samples taken before, by taking (25) gram from each sample then each sample was mixed in (225) ml of distil water followed by diluting them to the second dilution. Again (1) ml from all sample in Petri dish and added media. The next step was all samples were put in an incubator (30ºC) for 5 days, then the result were written by the isolate of mould.

• 2.1.2.1. Medium:

The medium of choice was sabouraud dextrose agar according to FAO (1992). As described by speck (1976) directions and sterilized. Then the medium was tempered to 45ºC, and 2ml of the antibiotic solution per 100ml of the medium were added and mixed.

• 2.1.2.2.Sabouraud dextrose agar: -

(9.8) gram of agar powder was weighted (150) ml of distil water was added then put in to the autoclave in a temperature of 121 ºC for 15 min, and atmospheric pressure of (15) pound| inch for quarter, for sterilization.

• 2.1.2.3. Antibiotic Solution:

This was made according to Leininger (1976) 500 mg each of chlortetracycline and Chloramphenicol were added to 100 ml sterile phosphate buffered distilled water and mixed. Two ml of this solution was added per 100 ml of tempered agar giving a final concentration in the medium of 100 mg/L of each of the antibiotics. After swirling, the medium is ready for use.

2.1.3.Examination of Mould :-

• Recorded the colonial characteristics and examine the colonies under the x10 (low power) of the microscope.

• Prepared slides of the mould growth for microscopic examination in the following way.

-Put part of sample on clean slide by loop.

-Added (2) drop of KOH (20%).

-Put cover glass on the slide, taking care to exclude air bubbles and flamed.

-Sample microscopic used lenses (x10) and then used the x40 (high power). (safe Alden, alzrwg 1993)

2.1.4.Examination of Yeast :-

• Colonial characteristics.

• Prepared wet mounts by suspended a protein of culture in a drop of water .added a small

drop of Gram’s iodine and cover with cover class .Observed with the x40 (high power) of

The microscope.

2.1.5.Identification of Mould and Yeast:-

Moulds are identified on the basis of morphological and culture characteristics including :-

• The Colonial characteristics:-size, sureface, appearance, texture, and colour of the colony.

• The vegetative mycelium:-presence or absence of cross-walls, and diameter of hyphae.

• The asexual and sexual reproductive structures :-( e.g. sporangia, conidial heads, zygospores, and arthrospores).

RESULTS

A number of mould genera were identified in the present investigation. The most dominant was Aspergillus sp and Rhizopus. 45% of all cheese was found contaminated by fungi. Mould species isolated from cheese samples were Aspergillus sp35%. Rhizopus sp 10 %. The yeasts genera isolated from the cheese samples were also identified. The most dominant was Debaryomayces and Saccharymyces. 55% of cheese samples were contaminated by yeasts species, (Debaryomayces 35% and Saccharymyces 20 %).

• Minimum and maximum range of mould contamination in cheese 6x102 - 7x104 g | ml respectively compare with acceptable range Sudanese Standers Metrology Organization (ssmo) shown in Table (3.1).

3.1.Yeasts and moulds counts (CFU/g):

• The total viable count for microorganism contaminating of yeast and mould was: 1x102---- 9 x102, 1x102---- 2x102 CFU/g respectively is shown in Table and table (3.2) and (3.3). • The Microscopic colonial morphology and mould species shown in figures (3.1), (3.2), (3.6), (3.7), (3.8) (3.12), (3.13) and (3.14). • Yeast species shown in figures (3.3), (3.4), (3.5), (3.9), (3.10) and (3.11).

Table 3-1 Minimum and maximum range of mould contamination in cheese compare with acceptable Sudanese Standers Metrology Organization (ssmo).

Product Presence Acceptable (SSMO) Range of Mould g|ml

Minimum Maximum Minimum Maximum

White cheese 6 x 102 7 x 104 Nil Nil

Table 3.2 Total viable counts (T.V.C) of yeasts isolated from 100 samples of cheese collected from different retail sale in Khartoum North.

Yeast Sample Code No .of colony |g

saccharomyces 1 1x102 2 5x102 3 3x102 4 2x102 5 5x102 6 3x102 7 2x102 8 1x102 9 6x102 10 4x102 11 3x102 12 3x102 13 3x102 14 3x102 15 1x102 16 6x102 17 2x102 18 1x102 19 1x102

20 1x102

Total 20 2.8x102 CFU|g

Debaryomayces 1 6x102 2 1x102 3 un countable 4 un countable 5 un countable 6 un countable 7 un countable 8 un countable 9 un countable 10 4 x102 11 9x102 12 7x102 13 2x102 14 5x102 15 4x102 16 3x102

17 1x102 18 8x102 19 6x102 20 1x102 21 3x102 22 8x102

Yeast Sample Code No .of colony |g

Debaryomayces

23 3x102 24 5x102 25 9x102 26 2x102 27 3x102 28 1x102 29 2x102 30 2x102 31 4x102 32 1x102 33 1x102 34 2x102 35 6x102

Total 35 7.4x102 CFU|g

Table 3- 3Total viable count (T.V.C) of moulds isolated from 100 samples of cheese collected from different retail sale in Khartoum North.

Mould Sample Code No .of colony |g Rhizopus

1 3x102 2 3x102 3 3x102 4 3x102 5 2x102 6 2x102 7 1x102 8 1x102 9 1x102 10 1x102

Total 10 2x102 CFU|g

Aspergillus 1 1x102 2 1x102 3 1x102 4 1x102 5 1x102 6 1x102 7 1x102 8 1x102 9 1x102 10 1x102 11 1x102 12 1x102 13 2x102 14 2x102 15 2x102 16 2x102 17 1x102 18 1x102 19 1x102 20 1x102 21 1x102 22 1x102 23 1x102 24 1x102 25 1x102

26 1x102 27 1x102 28 1x102 29 1x102 30 2x102 31 2x102 32 2x102 33 1x102 34 1x102 35 1x102

35 1.2x102 CFU|g

Figures (3.1)

Slides culture of Aspergillus pink colony on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail sale in Khartoum North.

Figures (3.2)

Slides culture of Aspergillus fumigats green and dark colony on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail saleKhartoum North.

Figures (3.3)

Slides culture of saccharomyces white colony on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail saleKhartoum north.

Figures (3.4) Slides culture of Debaryomayces stained dark on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail sale Khartoum north 34

Figures (3.5) Slides culture of Debaryomayces white colony on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail sale Khartoum north

Figures (3.6)

Slides culture of Aspergillus niger black colony on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail sale in Khartoum North.

Figures (3.7)

Slides culture of Rhizopus dark pink on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail sale in Khartoum North.

Figures (3.8)

Slides culture of Aspergillus fumigats green and dark colony on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail saleKhartoum North.

Figures (3.9) Slides culture of Debaryomayces stained dark on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail sale Khartoum north

Figures (3.10)

Slides culture of saccharomyces white colony on sabouruad Dextrose Agar isolated from 100 samples of cheese Collected from different retail saleKhartoum north.

Figures (3.11)

Yeast colonies on sabouruad Dextrose Agar Isolated from 100 samples of cheese collected from different retail sale in Khartoum North.

Figures (3.12)

Aspergillus sp colonies on sabouruad Dextrose Agar isolated from 100 samples of cheese

Collected from different retail sale in Khartoum North.

Figures (3.13)

Aspergillus niger on Sabouraud dextrose agar white colony producing brownish dark conidia Isolated from 100 samples of cheese collected from different relail sale in Khartoum North.

Figures (3.14)

Aspergillus fumigats on sabouraud dextrose agar pink colony Agar Isolated from 100 samples of cheese collected from different relail sale in Khartoum North.

. DISCUSSION

The study was conducted in Khartoum North locality to identified types of fungi encountered in cheese at Khartoum North markets. The results of this study showed different levels of contamination by mould and yeast. Yeasts and moulds present a problem in foods in that they discolor food surfaces, cause off-odors and off-flavors, because various degrees of spoilage alter substrates allowing for the outgrowth of pathogenic bacteria, and are able to produce mycotoxins in certain instances. The quality of cheese is determined by its flavor, texture, appearance and presence or absence of moulds. The quality of cheese is influenced by its composition, especially moisture content, NaCl concentration, pH, and moisture in nonfat substances and percentage fat in dry matter (Gilles, 1973). The growth of mould on the surface (e.g. Brie and Camembert) or interior (Blue varieties), or the growth of microorganisms on the surface (smear-ripened cheeses).Since the changes in appearance are visually perceptible, they are the criteria by which the consumer initially judges cheese quality and hence are of major significance. These changes are not just cosmetic they are visual evidence that the flavor and texture are satisfactory. The absence of mould in mould-ripened cheese clearly indicates unaccepted able quality, and of course the growth of moulds on non mould varieties indicates spoilage. Traditionally, the surface of cheese was ex posed to the atmosphere, and hence loss of moisture occurred. The loss of moisture is especially critical for varieties in which the growth of microorganisms on the surface is a key feature of ripening, and thus such varieties are ripened in high-humidity environments, traditionally in caves with naturally high humidity and frequently now in environments with artificially controlled humidity (Lawrence, 1984).

The texture of cheese has a major impact on flavor perception. For example, it has been suggested that the main contribution of proteolysis to cheese flavor is due to its effect on cheese texture, which affects the release of sapid compounds during mastication of the cheese. There- fore, these attributes should be considered together. However, they rarely are, and cheese texture is even less well understood at the molecular level than cheese flavor (Olson, 1990).

Cheese was found contaminated by fungi, among the moulds important in food spoilage are species of Asperigillus and Rhizopus of this Aspergillus has aroused concern because some species contain strains capable of releasing toxins in food. Some of these toxins are carcinogenic and possible association of incidence in liver disease with consumption of food materials contaminated with mould toxins (Longree, 1980). In the present studies the incidence of Aspergillus in the sampled cheese which presented a potential risk of mouldy cheese for consumers. Many fungi naturally produce a substance known as a mycotoxin during their digestive process. These mycotoxins are toxic to humans, and some are extremely toxic if ingested even in small quantities. There are some foods that naturally contain high levels of these mycotoxins, and most of us aren't even aware that mycotoxins exist, much less that we eat them in our food supply. The foods that most often contain high concentrations of mycotoxins are grain crops and cheese. The fungi can grow through the material. Here again, there is direct consumption of the fungi that lives in the cheese (James, 2005)

Most fungi-produced mycotoxins are harmless, and even helpful. For example, the antibiotic penicillin came from a fungus, and it is a mycotoxin. Some of these fungi (primarily Aspergillus sp) produce the very lethal mycotoxins called aflatoxins. Aflatoxins are remarkably potent, often causing disease even when ingested in minute amounts. Aflatoxins can cause disease throughout the body, but are most commonly known for causing acute or chronic liver disease and liver cancer. This liver toxicity can produce a cumulative effect over time, and can eventually lead to diseases of the liver including hepatic fibrosis, cirrhosis, and fatty liver disease. Aflatoxins are also carcinogenic, causing a variety of different cancers. They can cause blood disorders where small arteries are blocked due to blood clots (Davis, 1968)

Also cheese was contaminated by yeasts species, Debaryomayces and Saccharymyces Dirar (1975) stated that under tropical conditions many factors such as high temperature, absence of sanitary conditions for production of milk in the dairy farms and unavailability of cooling during handling and transportation of milk affect the quality of milk.

The factors controlling the growth of microorganisms in cheese include water activity, concentration of salt, oxidation reduction potential, pH and ripening temperature.

CONCLUSSIONS

The study concluded that cheeses were heavily contaminated by different types of moulds and yeasts, this indicates that cheese was produced under non hygienic conditions or storage and handling. According to that a number of recommendations from the Good Manufacturing Bract ice (G.M.P) and Hazard Analysis and Critical Control (HACCP)-as Stated by the Codex Alimentarius –were stated to improve the hygienic state of cheese in Khartoum North locality.

RECOMMENDATIONS

1. Establishment or development of milk / milk product hygiene program. 2. Improvement of cheese quality by introduction of simple hygienic principles for cheese production and handling, by applying the Good Manufacturing practice (G.M.P) (Hazard Analysis and Critical Control (HACCP). 3. Following food legislation. 4. Development of standards and grades for raw milk. 5. Proper and efficient sterilization for milk need to be adopted if it is desired to obtain a high quality cheese product hygienic.

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ﺳﻴﻒ اﻟﺪﻳﻦ اﺣﻤﺪ ﺟﻤﻴﻞ ،اﻟﺰروق ﻣﺼﺒﺎح اﻟﺴﻨﻮﺳﻰ ،(1994).اﻟﺪراﺳﺔ اﻟﻌﻤﻠﻴﺔ ﻟﻠﺒﻜﺘﺮﻳﺎ واﻟﻔﻄﺮﻳﺎت اﻟﻄﺒﻴﺔ.اﻟﺪار اﻟﻌﺮﺑﻴﺔ ﻟﻠﻨﺸﺮ واﻟﺘﻮزﻳﻊ .اﻟﻄﺒﻌﺔ اﻻوﻟﻰ 1993.

University of Khartoum

Graduate collage

Medical and Health Studies Board

Types of Fungi Encountered in Cheese at Khartoum North Markets

Hawyda Abdel Azim Abd Alatif Mohammed

(B.P.H) - of.Shendi

A thesis submitted in partial fulfillment for the requirements of the degree of master in Public and Environmental health (Food hygiene and safety)

Supervisor:

Dr/ Elrasheed A.Ali

B.Vet.Sc-M.Sc-Ph.d

Khartoum -2010