Isolation and Identification of Rhizopus From Decaying Bread
CHAPTER ONE
AIM AND OBJECTIVES OF THE STUDY
This project work is based on the Isolation and Identification of the fungal organism responsible for the spoilage of Bread. It is also to determine the rate of growth of the Organisms and to know its ability to grow on inhibitory media. To examine the species of fungi that infests breads at room temperature.
CHAPTER TWO
LITERATURE REVIEW
COMPOSITION OF BREAD AND IT’S REQUIREMENT
Bread has become an essential part in Nigerian diet principally because of its ready availability and relatively cheap cost compared to other bakery products. Nearly all the commercial bread available in Nigeria are white in colour, sweet in taste and spongy in texture and usually produced by the straight dough method. Bread from the nutritionist perspective may be defined as the product obtained by doughing fermenting and baking of wheat flour with or without other ingredients (food standard committee report 1960). Flour is characterized by relatively low protein and higher carbohydrates content and the carbohydrate sugars of which 90% of more starch. Although, not an outstanding good source of most nutrients (frazier and westhof, 1986).
Enrichment of bread with vitamins and minerals have been instrumental in improving the nutritional adequacy of our diet, supplementing bread with protein concentrates and pure amino acids like “Lysine” shows great promise improving the diet of people who depends on bread as the main source of nutrient (Uddoh, 1980).
SOURCE OF CONTAMINATION
The heat baking kills all fungal spores that may be in flours or other ingredients of the bake goods, but break and other bakery products are subject to contamination from air borne fungal spores, as soon as baker has a high population of fungi, the bread almost inevitably becomes inoculated with a number of spores before it leaves the bakery source. Contamination in bread can also be from handling of the wrappers and usually initiate growth in the middle of the load and between the slices of sliced bread. The most important thing, if our bodies are to obtain the full nutritional values of the food eaten. Since contaminated food will lead to irritation of the gastrointestinal track Infection (disease leading to inevitable loss of nutrients (Uddoh, 1980).
RHIZOPUS AS A SPOILAGE ORGANISM
When the term spoilage is applied to foods, decay or decomposition of undesirable nature is usually implied. This is caused by growth and activities of microorganism such as rhizopus. Rhizopus is the most common and hence the most important cause of the spoilage of bread. It can cause zygoycoses and other infections in compromised individuals. In fact, most of bakery product moldiness was common in tropics and it occurs within loaf whenever moisture is available. The filamentous types of fungi cause bread moldiness in the topics and expansion of these species were found to be between 3.4 and 5.5 (ingold, 1973). Fungi already reported to be involved in bread moldiness include rhizopus nigricans, popularly known as cotton mycelium with black dots of sporangia i.e “black mould” or “breed mould”, which causes rhizopus soft rot, the green spored caused by pencillium expanum or p. stoloniferm, aspergillus niger with its greenish or purplish, brown to black conidial head and yellow pigment diffusing into the bread (Christensen, 1965). The yellow mould of Aspergillus Oryzea is some times found while Odium auanticum produces orange spots on bread (Bonner, 1967).
CHAPTER THREE
METHODOLOGY
MATERIALS AND METHODS
SAMPLE COLLECTION
Samples of bread to be collected from bakeries in Enugu town, taken to the laboratory immediately for analysis, is to be left at room temperature for 8 days so as to allow visible growth of fungal organisms (filamentous fungi) which will be used later during culturing.
PREPARATION OF MEDIUM
The medium to be used is nutrient agar. The composition of the medium as follows:
Beef extract 30gramms
Yeast extract 2.0g
Peptone 5.0g
Nacl 5.0g
Agar (oxoid) 20.0g
Distilled water 1000ml
PH 7.0
DIRECTION FOR METHOD OF PRIPARATION OF THE MEDIUM
5.6 grammes of nutrient agar (NA) are to be weighed into 200ml of distilled water in a conical flask. This will then be mixed gently by shaking, and then cocked with cotton wool and foil paper; it will also be sterilized in the autoclave at 1210C for 5minutes. After autoclaving, 0.2ml of streptomycin will then be added to inhibit bacterial growth.
MATERIALS TO BE USED FOR THE PREPARATION OF THE MEDIUM
These includes glass wares such as:
- Petridishes (2)
- Test tubes (5)
- Measuring cylinder
- Beaker.
These will be washed and rinsed thoroughly with distilled water. They will then be sterilized in the hot air oven for 2 hours at 1600C.
Inoculating loops, Needle, Knife.
All these are to be sterilized by red heat using spirit lamp, dipped into methylated spirit and dried to cool before use.
CHAPTER FOUR
GENERAL MORPHOLOGY
GENERAL MORPHOLOGY OF RHIZOPUS
Rhizopus forms a mass of soft, closely woven white, silky threads. This mass, which is known as a mycelium, is the vegetative part of the fungus. Each silky thread like structure is a hypha.
The mycelium consists of three sorts of hyphae, they are stolons which grow horizontally on the substrate, rhizoids or root like hyphea which arise at points where the stolons come into contact with the substrate, and sporangiophores. The rhizoids are much-branched hyphae, which penetrate into the substrate. They are able to digest and absorb organic food. The sporangiophores are erect unbranched hyphae arising from the stolons at the same parts where rhizoids are formed. They grow vertically upwards and gives rise to the reproductive structure called sporangia.
Each hypha is non septate, i.e it has no cross walls dividing it up into cells. Its wall is composed of proteins and carbohydrates. The interior of each hypha contains granular cytoplasm in which lie numerous nuclei. The hypha is, therefore, described as multinucleate. The cytoplasm also contains abundant reserve food substances in the form of oil droplets, vacuoles of varying sizes and granules of glycogen.
CHAPTER SIX
CONCLUSION AND RECOMMENDATION
CONCLUSION
It has been observed that most of the food materials like garri, yam and bread are being spoiled by rhizopus. It has also been noted that rhizopus are generally aerobic plants whose growth has facilitated by little moisture and that the best growth was from the one cultivated for four (4) days.
When the organisms were inoculated in nutrient agar and incubated for four (4) days, it was found that the organisms has over grown and had already been sporulated, showing that 4 days is too much for incubation. When the plate were incubated for two (2) days, the growth occurred but not well pronounced but on incubating the plate for three (3) days at 37oC, the organisms grow very well and all the parts are seen. This shows that the best or maximum period for incubation of mould is three days.
Again when isolating muoulds, one has to look for the best media for the growth. These media have to be carefully prepared to avoid contamination.
In the experiment, nutrient agar was carefully prepared and poured in a sterilized petri-dishes. The organisms were inoculated using a sterilized wire-loop. Even the slide on which the colony was placed before mounting on a microscope was carefully sterilized.
Juging from the result, rhizopus sp was isolated indicating that the culture was pure. Therefore the aim (to determine the rate of growth of the organism) was achieved.
RECOMMENDATION
Since it is known that after three (3) days of baking bread, there will be infestation which leads to growth of rhizopus on the product (bread), we thereby recommended that NAFDAC should inspect all the bakery industries to ensure that the manufacturing and expiration date of the bread is written on the label before distributing, so that the basis of selling well-baked breads/uncontaminated breads will be employed in order to avoid any sort of food poising associated with ingestion of bread.
REFERENCES
- Bonner, J. T. (1967): Fundamentals of Mycology. London pg. 205.
- Brul, S. and P. Coot (1999): “Preservative Agents in Food mode of Action and Microbial in Resistance Mechanism”. International Journal of Food Microbiology Vol. 5, No. 3, Oxford University Press; pg. 5.
- Christensen, C. M. (1965): The moulds and Man. 3rd edition, Minneapolis, pg. 284.
- De Boer, E. and R. R. Beumer (1999): “Methodology for Detection and Typing of Food Borne Microorganism”. International Journal Food Microbiology: Vol. 5, No. 3, Ozford University Press, Pg. 23 – 30.
- Frazier, W. C. and d. C. Westhoff (1986): Food Microbiology. 3rd edition, 6th reprint, Tata Mogrew-Hill Publishing Company Limited, New Delhi, Pg. 208 – 209.
- Jay, J. M. (1996): Modern Food Microbiology. 5th edition, New York, Chapman and Hall pg. 28 – 30.