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The fungi kingdom is one of the biggest group of microorganism that related with food, this group can be divided into tow group mould and yeast, mould have wide verity of species from the big mould like mushroom to the microscopic one like penicillium these organism consist of multicellular filaments and they have the ability to grow in very low aw and acidic condition, also what make them unique they produce big numbers of asexual spores, which has dry thick wall ,and pigment to protect them from the harsh condition, and then it can be carry by the air to long distances this made mould wide spread in many type of food like cereal ,nuts ,fruits ,vegetables and it can spoil these food or it can produce mycotoxin on it , the old method to detect mould was based on bacterial detection by keeping the plat for long time and test the filamentous under microscope, then they start using acidified media or by adding antibiotics to the media for example potato dextrose agar with PH 3.5 , whereas the yeast have slightly different characteristic it is unicellular fungi and it reproduce by budding or fission .The cell morphology like type of cell division and the shape of the spores are used to identify the different species, there are around 900 species of yeasts and only around ten species conceder as spoilage organism , most yeast prefer to grow in food with low PH around 5.5 or lower , good amount of sugar, organic acids and it usually found on the surface of products like cheeses and meats. However yeast has many applications in food industry it involves in many products like bakery products, and in making beer.
The genus Saccharomyces cerevisiae this name mean sugar fungus it is really common in these type of products and it produce commercially in wide rang, in our experiment we will test sample from flour and a sample from dough that have been made fro the same flour.
Sample of flour and dough
Standard yeast dough sample
1 bottle Malt extract agar
Plates of Oxytetracylin-Glucose-Yeast-Extract agar (OGYE)
Dilution tubes (9 ml Peptone Saline, PS)
Dilution bottles (90 ml Peptone Saline, PS)
Beaker with ethanol
Stomacher and stomacher bags (sterile)
Incubators set at 30°C
Petrifilm Yeast and Mould Count Plates (3MJ)
A) Making the Bread
Mix sugar and salt together
Add the yeast
Add water and keep it for 15 min to let the dried yeast rehydrate
Add most of the flour and mix until the texture is coherent and elastic
Left the dough to rise in a warm place at 35°C for about 1-2 h
Then kneading again the dough is now left to rise for about 30 min and subsequently baked for 30 min at 200°C.
b) Sampling preparation
Take 10 g of the flour and dough from the package into a separate stomacher bag.
Add 90 ml of sterile Peptone Saline (PS) solution to the sample and homogenized in the stomacher for 1 to 2 min, and this will be the first dilution.
Make further dilutions 10-2,10-3 ,10-4 ,10-5 ,10-6 ,10-7 ,10-8
c) Enumeration of yeasts and moulds.
Prepare 7 additional dilutions from the stomacher bags for the dough sample.
Prepare 2 additional dilutions for flour sample.
Plate three consecutive dilutions For each sample
Pipette 0.1 ml diluted sample10-5 ,10-6 ,10-7 for dough and 10-1,10-2,10-3 for flour onto the OGYE agar and spread it carefully with the flamed glass spreader.
Pipette 1.0 ml diluted sample10-6 ,10-7 ,10-8 for dough and 10-1,10-2,10-3 for flour onto an empty Petri dish and add ME agar.
Do not invert the plates and incubate aerobically at 30°C for 5 days.
d) Enumeration of yeasts and moulds on 3MJ Perifilim
Pipette 1.0 ml diluted sample onto one dehydrated Petrifilm. For the dough sample plate 10-6 ,10-7 , and 10-8
evenly distribute the sample according to the instructions using spreader
Incubate the Petrifilm plates with the clear side up in stacks for 5 days at room temperature (21-25°C).
e) Microscopy of reconstituted dried baker=s yeast.
Dissolve 1 teaspoon yeasts in hand warm water in a beaker (250 ml). Make a wet mount on a microscopy slide and add the cover slip. Now look under the microscope using the x 40 ocular.
Table 1.dough on OGYE agar (spread plating)
Table 2.dough on ME agar (spread plating)
Table 3.flour on OGYE agar (spread plating)
Table 4.flour on ME agar (spread plating)
Table 5.dough on 3M petrifilm
The morphology of Saccharomyces cerevisiae under the microscope is big oval colonies non motile, with size around 4µm
In comparing the results that we got for the dough in the tow type of media it seam like the ME media work better for enumeration of yeast and mould that because the number of yeast and mould was slightly higher than the OGYE media and from table 1 and 2 the number are 6 x108 Cfu/g , 6.3x 107 Cfu/g respectively. By looking to the table 3 and 4 which show the number of yeast and mould in the flour sample in tow different medium, it is very obvious there are big difference between the count for dough and flour, dough has greater number of yeast and mould that because of the higher aw , and more nutrient like suger that we added during making the dough ,also it important to bear in mind the number yeast which added with mixture this for sure will increase the total number of yeast and mould in the sample.
From table 5 which shows the number of yeast and mould for dough sample in different type of media it is 3M petrifilim which dehydrated media which is ready to use media and more easy to work with, it seam like it provide us with more accurate result that might be due to the counting grid which will help counting even the small colonies.
1. What roles do yeasts play in the making of leavened bread?
1-yeast will ferment sugars and this will release co2 and ethanol which will form babble in the dough and this will make the beaked product lighter, more easy to chew.
2. Are yeasts growing aerobically or anaerobically in the dough?
2- it grow in anaerobic condition.
3. Most baker=s yeast comes from distilleries where the S. cerevisiae is used to produce liquor (ethanol). Does bread contain any amounts of ethanol?
3- No, most of the ethanol will volatize during baking.
4. How does the results from the three different methods for enumeration of yeasts and Moulds compare? Did you find the Petrifilm from 3M easy to use, why or why not?
4- It gave slightly higher count, yes using the 3M petrifilm much easier there is no need to prepare the media, it counting grid helps in counting, save space in the incubation , it can reduce the amount of waste.