Etiket Arşivleri: FOOD MICROBIOLOGY
Indicators in Food Microbiology
Bacterial Groups Relevant to Food Microbiology
In Unit 1 we classified bacteria accordingly:
Very Bad (Pathogenic)
The primary interest in food microbiology is producing safe food with adequate shelf life
Looking for specific bacteria that cause spoilage or food-borne illness is like looking for a needle in a haystack
An indicator (index) in food microbiology is needed to confirm that the food is safe and has adequate shelf life
• Provide a gauge of product shelf life
• Highlight potential hazards
• An assessment of the previous history of food product
• Evaluation of the efficacy of control measures to prevent and/or inactivate microbial activity
Types of the bacterial counts will depend on the nature of the product
• Total Aerobic Count
• Psychrotrophic Count
• Lactic acid bacteria
• Yeast and molds
FE 421 FOOD MICROBIOLOGY LABORATORY
Name of student : M. Hakan MAVİŞ
Group : B – 2
Name of experiment : Milk and Milk products, Yogurt Analysis
The purpose pf this experiment was to analyze microbiological properties of yogurt and to investigate the number of lactic acid bacteria and formation of mold and yeast.
The effect of yogurt as a dietary supplement was investigated with regard to the gut ecosystem and lipid metabolism of 12 healthy, elderly people (78.3 +/- 9.8 years, body mass index 23.6 +/- 5.3 kg m -2, and mean +/- SD). Commercial yogurt with homogenized fruit was prepared by fermenting milk with yogurt specific cultures Lactobacillus delbrueckii ssp. bulgaricus (strain AY/CSL) and Streptococcus thermophilus (strain 9Y/CSL). The subjects consumed their usual diet (equal to 6279-6698 kJ d -1) over a 2-week baseline period (baseline start to end) and then were supplemented for 4 weeks with 250 g d -1 of fruit yogurt. The yogurt was administered in 125 g portions twice per day: at breakfast in substitution of milk and in the afternoon in substitution of tea with milk (test). At the end of the 4-week period the volunteers returned to their usual diet for a further 4 weeks (follow-up). At the end of each trial period no changes were observed in faecal water content, pH, bile acid concentration or cytolytic activity of the faecal water. Throughout the study there was significant variation neither in dietary intake of macro- and micronutrients, nor in the plasma lipids and, during the experimental period, in the counts of the total anaerobic microorganisms, bifidobacteria, lactobacilli, coliforms or enterococci. The only significant difference was observed in the clostridia counts, which decreased (P < 0.05) after the consumption of yogurt. Moreover, this effect was still evident at the end of the follow-up period. Since this last result can be considered a positive modification of the colon ecosystem, as clostridia are involved in the production of putrefactive compounds, it is possible that a yogurt-supplemented diet can maintain and/or improve the intestinal microbiota of elderly subjects.
a) Total Count:
Firstly; from yogurt dilutions were prepared from 10-1 to 10-6 dilutions. For this, 25 g yogurt sample was weighed and added above 225 ml peptone water thus 10-1 yogurt dilution was prepared then; from 10-1 dilution 1ml was taken and it was added to 9 ml water thus 10-2 dilution was prepared. In the same manner, 10-3, 10-4, 10-5 and 10-6 dilutions were prepared. Finally; from each tube 0, 2 ml was taken with pipette and sample dilutions were put on the plate count agar (PCA) and then spread out with spread plate method and incubated at 30 oC for 1 to 3 days. After incubation number of microorganisms in gram was calculated in yogurt.
b) Lactic Acid Bacteria:
At previous experiment prepared dilutions were used for this experiment. Again sample dilutions were taken 0, 2 ml and put on the mean rogosa sharp agar (MRS). Then; plates were incubated at 35 oC for 24 to 48 hours. Finally; number of lactic acid bacteria was calculated in gram.
c) Mold and Yeast Count:
Again, at first experiment, preparaed dilutions were used for this analysis. In here; 0, 2 ml sample dilution was taken and was put on potato dextrose agar (PDA) and spread out with spread plate method. Then PDA was incubated at 25 oC for 2 to 5 days. Finally; number of microorganisms was calculated in gram.
Plate count agar
Mean rogosa sharp agar
Potato dextrose agar
Peptone water pipette
Test tube rack
RESULT and CALCULATIONS:
Mold & Yeast
Number of microorganisms = (46 * 101) / 0, 2 ml = 2300
Number of microorganisms = (198 * 104) / 0, 2 ml = 9, 9*106
Number of microorganisms = (189 * 104) / 0, 2 ml = 9, 45*106
Average number of m / o’s = (9, 9*106 + 9, 45*106) / 2 = 9, 675106
Number of microorganisms = (152 * 104) / 0, 2 ml = 7, 6*106 (pasteurized)
Number of microorganisms = (207 * 104) / 0, 2 ml = 1, 035*107 (raw)
In this experiment, we examined properties of microorganisms and calculated the number of microorganisms in yogurt. For this, we made three analyses. For the number of microorganisms on plate count agar, for lactic acid bacteria on mean rogosa sharp agar and for mold and yeast on potato dextrose agar were used for yogurt microbiologic analyses. In here; MRS was used to determine total number of lactic acid bacteria. PDA was used to determine the number of mold and yeast. When we diluted the yogurt sample, we used the peptone water has a great protective effect. For this, 1g peptone was dissolved in 1L of distilled water and pH was adjusted to 7, 0 and it dispenses in sufficient quantity to allow for loss during sterilization.
The end of experiment, at pasteurized yogurt 2300 microorganisms was calculated on PCA. Actually; in open yogurt more microorganisms should have been observed. On the other hand; also in here, these microorganisms were useful microorganisms like Lactobacillus bulgaricus and Streptococcus thermophilus.
On MRS, lactic acid bacteria were calculated as 7, 6*106 for pasteurized yogurt and 1, 035*107 for open yogurt.
FE 421 FOOD MICROBIOLOGY LABORATORY
Name of student : M. Hakan MAVİŞ
Group : B – 2
Name of experiment : Seafood
The purpose of this experiment was to investigate microbiological affectivities of fish.
International competitiveness requires optimal productivity, quality and value, and the development of new products from traditional raw materials, underutilized species and waste streams. The productivity and competitiveness of seafood processing depends not only on the sources and costs of raw materials, but also on other costly resources: energy, water, labor and waterfront space. Energy equipment for thermal operations (refrigeration, cooking and retorting) is tremendous, yet opportunities exist for conservation through energy and water audits and demonstrating new technologies at processing plants. Solid waste disposal is a mounting problem for the industry as coastal populations and environmental sensitivities increase. This problem by developing enzymatic and microbial methods of hydrolysate manufacture for feed and fertilizer production, and improving manufacturing methods and uses of dried meals.
Seafood is among the most expensive items in the American diet due to the high costs of catching, transporting, processing and storing this delicate commodity. Although profit margins are small, improved post-harvest technologies offer opportunities to increase product quality and profits. Seafood muscle tissues are the most valuable component of seafood products—they have many desirable properties due to their water- and fat-binding traits, which can be enhanced by non-seafood additives and novel processing techniques. Ready-to-cook and ready-to-eat seafood products require processing and storage that can reduce product quality. A better understanding of the chemical and physical properties of seafood muscle components could minimize these effects. Many fish species are not widely consumed for food because they degrade rapidly. Improved storage and processing techniques would help; but because fish and shellfish are highly variable in their physiology, their properties need to be studied by species. New enzymes, enzyme inhibitors and other “active” proteins, such as antifreeze proteins, could be isolated from seafood sources and used to add value to other seafood.
PCA (plate count agar)
Test tube rack
Firstly; a sterile swab was taken and it was plunged into 0,1 % peptone water for soaking then swab was spread on surface of fish about 10 cm2, swab was put into 10 ml 0,1 % peptone water after breaking tip off of swab then. Swab and peptone water were shaken then 0,5 ml sample was taken from tube containing swab and peptone water and inoculated to PCA and incubated at 37 oC for 24 hours.
Secondly; coliform test was applied on fish. 0,5 ml sample was taken and inoculated to violet red bile agar (VRBA) and then incubated at 37 oC for 48 hours. Finally; number of microorganisms was calculated in 1 cm2.
RESULTS and CALCULATIONS:
These values were in 10cm2 and for 1cm2:
# of m/o’ s = 216*1 cm2 / 10cm2 = 22
# of m/o’ s = 177*1 cm2 / 10cm2 = 18
# of m/o’ s = 244*1 cm2 / 10cm2 = 24
# of m/o’ s = 152*1 cm2 / 10cm2 = 15
# of m/o’ s = 202*1 cm2 / 10cm2 = 20
# of m/o’ s = 200*1 cm2 / 10cm2 = 20
# of m/o’ s = 147*1 cm2 / 10cm2 = 15
# of m/o’ s = 90*1 cm2 / 10cm2 = 9
# of m/o’ s = 110*1 cm2 / 10cm2 = 11
# of m/o’ s = 11*1 cm2 / 10cm2 = 1
# of m/o’ s = 7*1 cm2 / 10cm2 = 1
# of m/o’ s = 15*1 cm2 / 10cm2 = 2
# of m/o’ s = 40*1 cm2 / 10cm2 = 4
# of m/o’ s = 24*1 cm2 / 10cm2 = 2
# of m/o’ s = 108*1 cm2 / 10cm2 = 11
# of m/o’ s = 17*1 cm2 / 10cm2 = 2
# of m/o’ s = 7*1 cm2 / 10cm2 = 1
# of m/o’ s = 5*1 cm2 / 10cm2 = 1
In this experiment we studied the seafood and for this we used a fish, and at 10cm2 number of microorganisms were examined. In order to analyze we used swab. Swab is a sterile loop. Swab was spread the surface of fish then heat part or cotton part was broken down so as to not touch our hand, because on our hand some microorganisms may be and these microorganisms with swab together can be put in 10 ml sterile water. If it is so, our results can be false so we must care when this process was applied.
Living fish carries gram negative psychrotropic bacteria on their surface and also fresh fish carries 102 or 103 bacteria per 1cm2 on surface, also our result shows in results and calculation part. Stale fish can be to include more microorganisms and these microorganisms can harm to human health.
FE 421 FOOD MICROBIOLOGY LABORATORY
Name of student : M. Hakan MAVİŞ
Group : B – 2
Name of experiment : Methylene Blue Reduction Test and Direct Microscopic Count Method
The purpose of this experiment was to investigate the coliform in milk and learn methylene blue reduction test and direct microscopic count method for milk.
The Standard Plate Count (SPC) procedure is used to determine the number of bacteria in a sample. In most cases the initial day SPC represents those bacteria that survive pasteurization (thermoduric), although gross contamination after pasteurization can cause high counts. The regulatory standard of < 20,000/ml is generally easily achieved. Most initial day bacteria counts are <500/ml while counts higher than 1000/ml suggest a potential contamination problem, either in the raw milk supply or within the processing equipment.
The ideal milk shows no increase in bacteria counts during refrigerated storage. When milk is held under refrigeration, only bacteria capable of growth under these conditions will grow. While most bacteria prefer warmer temperatures for growth, some bacteria, referred to as psychrotrophs (“cold-loving”), are capable of growth at 45oF or less. The most common types of psychrotrophic bacteria that rapidly spoil milk do not survive pasteurization; thus their presence in milk is the result of post-pasteurization contaminants due to less than adequate sanitation practices. The initial day SPC of fresh pasteurized milk is not a good indicator of the numbers of psychrotrophs present since most bacteria that survive pasteurization are not psychrotrophic (a few types of thermoduric bacteria will grow slowly under refrigeration conditions). A significant increase in the SPC after 7-10 days of refrigeration storage is evidence of psychrotrophic growth and suggests that post-pasteurization contamination has occurred and that shelf-life will be shortened. Generally, when the SPC exceeds 1 – 100 million, the product will become unacceptable due to flavor defects related to bacterial growth. The key to preventing spoilage and extending the shelf-life of a product is to prevent post-pasteurization contamination through a well-designed quality assurance program. It only takes one psychrotrophic bacteria per container of milk to cause spoilage.
The coliform bacteria (coli) count is used as an index of sanitation during the handling and processing of milk products. Coliforms are killed by pasteurization, thus when present in milk, they are regarded as post-pasteurization contaminants resulting from poor sanitation. Though the standard is “not to exceed 10/ml,” detection of any coliform bacteria suggests that there is some point in processing that has been neglected in regard to effective cleaning and sanitation procedures. As a rule, the detection of coliforms in milk will indicate the potential for a shortened shelf-life due to concurrent contamination with psychrotrophic bacteria. Milks with coliform counts exceeding 10/ml are not tasted on subsequent days in this program.
Tcyptone glucose yeast agar
Methylene blue solution
Test tube rack
1 – Total Count:
Firstly; milk was diluted from non dilution milk to 10-5 dilution in the test tube. In order to make 1 ml non-dilution milk was taken and it was added to 9 ml distilled water and thus 10-1 milk dilution was occurred and this process was continued to 10-5 dilution. After that; 0, 2 ml dilution was taken with pipette from each dilution (non-dilution, 10-1, 10-2, 10-3, 10-4, 10-5) these were inoculated to tcyptone glucose yeast agar with spread plate method. Then; these were incubated at 37 oC for 24 hours. After incubation, between 300 – 30 being microorganisms were counted and in ml numbers of microorganisms were calculated.
2 – Methylene Blue Reduction Test:
Secondly; raw milk and pasteurized milk were studied separately. 10 ml milk was put in the test tube and 3 – 4 drops methylene blue solution was added to milk. Then; milk and methylene blue solution were mixed vigorously, then; these tubes were incubated at 37 oC for 30 minutes. Milk was examined and observed whether decolorization was occurred or not.
A – raw milk
B – paste. Milk
C – raw milk
Group A) For 10-2 dilution: (284*102) / 0, 2 ml = 142000
For 10-3 dilution: (65*103) / 0, 2 ml = 325000
For 10-4 dilution: (38*104) / 0, 2 ml = 1900000
(142000+325000+1900000) / 3 = 789000
Group B) For 10-1 dilution: (47*101) / 0, 2 ml = 2350
Group C) For 10-3 dilution: (198*103) / 0, 2 ml = 990000
For 10-4 dilution: (33*104) / 0, 2 ml =1650000
(990000+1650000) / 2 = 1320000
Methylene Blue Reduction à this method depends on the ability of microorganisms to change oxidation-reduction potential of medium. Bacteria consume dissolved oxygen in medium and produce some enzyme. These enzymes oxidize substrate and hydrogen removed from substrate and hydrogen was held with methylene blue solution and light blue of milk converted to white or colorless.
3 – Coliform Test:
Firstly; milk was diluted from non-dilution to 10-5 dilution, then 0, 2 ml diluted and milks were spread out on violet red bile agar with spread plate method then plates were incubated at 37 oC for 48 hours. After 48 hours number of colonies was counted and in ml number of microorganisms was calculated.
4 – Direct Microscopic Count Method:
In here; firstly; slide was taken and on slide 1 cm2 area was determined and one loopful raw milk was put on this area and raw milk was spread out with distilled water in this area. Then; on slide milk was waited to get dry in air. After that; one loopful xylol was added on each square and waited for 1 min then slide was washed with water. Afterwards; 1 – 2 drops methylene blue was added onto each square and waited for 2 min and again slide was washed with water. Next; slide was got dry in air. Finally; on each square, one drop iol immersion was added and milk was examined under 100X objective.
A – raw milk
B – raw Milk
C – raw milk
Group A) For 10-3 dilution: (165*103) / 0, 2 ml = 825000
Group C) For 10-3 dilution: (50*103) / 0, 2 ml = 250000
Direct Microscopic Count Method:
1st half slide
2nd half slide
1st half slide à 3+6+8+14+21 = 52
2nd half slide à 2+4+5+10+15 = 36
Average number of microorganisms = (52+36) / 10 = 8, 8
Average number of m/o’s = 8, 8 * 15700 * 100 = 14130000
In here 100 is 0, 01 ml milk.
In this experiment; we examined microbiological properties of milk. In milk number of microorganisms was calculated with total count method and coliform bacteria were investigated with coliform test. Also; methylene blue test was applied the milk. In this test one drop methylene blue solution was dropped into milk and colour of milk was slightly blue. After 30 minute slight blue colour of milk converted to white, this transformation was related with number of microorganisms in milk. Microorganisms were used the dissolved oxygen and microorganisms produced certain enzymes. These enzymes oxidize the substrate, thus hydrogen was removed from substrate. Hydrogen was held with methylene blue solution and color of milk was colourless this process was called decolourization. In addition; while raw milk decolorized for 30 minutes pasteurized milk decolorized for 4 – 5 hours. This showed that; in raw milk, number of microorganisms was more than in pasteurized milk.
Secondly; number of microorganisms was determined with total count method. In each milk dilution, number of microorganisms was calculated with accordance to between 30 – 300 microorganisms and as a result; we observed that raw milk was to contain more number of microorganisms than pasteurized milk.
Next; coliform test was applied the milk and in here number of microorganisms was calculated. Finally; in milk microorganisms was examined with microcopy and dark blue microorganisms were observed and at five regions average number of microorganisms were calculated, and these were determined at the result and calculation.
FE-206 Food Microbiology1
Microbial Growth – Kinetics First Order
First Order Kinetics
Food microbiology is concerned with all phases
Of microbial growth (lag,log, stationary, death phase).Growth curves are normally plotted as the number of cells on a log scale or log10 cell number versus time.
Table First order kinetics to describe exponential growth and inactivation
g can be calculated by
g=t/n=(0.3 t)/”log10N-log10N0 “
Example: Initial population is 103 CFU/ml and incerased to 106 cells in 300 min. What is generation time?
g=”0.3∗300″ /(6-3)=30 min or you can first calculate µ and then calculate g.
2.3log(N/N0)= µ t µ =0.023min-1 and g=0.693/ µ
µ can be obtained by slope of straight line when the log numbers of the cell is plotted against time.
Ex:Ground meat manufactured with N0=1.2*104 CFU/g.
How long it be held at 7°C before reaching a level of 108CFU/g (for µ=0.025 h-1)
Killing can be by heat, radiation,acid,bacteriocin and other lethal agents is also governed by first order kinetics.
D value=amount of time required to reduce N0 by 90% is the most frequently used constant.
The relationship between k and temperature is explained by arrhenius equation
Zvalue= a number of degees required to change in the D values by a factor 10, or
It is the temperature required for one log10 reduction in the D-value.
z-value is used to determine the time values with different D-values at different temperatures with its equation shown below:
where T is temperature in °F or °C.
This D-value is affected by pH of the product where low pH has faster D values on various foods. The D-value at an unknown temperature can be calculated  knowing the D-value at a given temperature provided the Z-value is known.
For example: If Dvaue at 121 °C is 1.5 min and z value is 10 °C. The D value at 131 °C will be 0.15 min.
Importance of Being small size
(Surface area)/volume=(4πr^2)/(4/3 πr^3 )=3/r high ratio
Cell mass is close to cell surface, no circulatory metabolism are required and this limits the size of bacteria to microscopic dimensions.
As the cell size increases, the s/v ratio decreases, which adversely affects the transport of nutrients into and end-products out of the cell.
Microbial Growth Characteristics in Foods
Energy and nutrient sources are often present in limiting concentrations; microorganisms compete each other for nutrients and results in exclusion of slower growing species.
Foods contain a mixed population of microorganisms. Competition among the different kinds of microorganisms in food determines which one will outgrow the others and cause its characteristics types of changes.
2. Metabiotic (Sequential) Growth
Different types of microorganism present normally in foods, but the predominant types can change with time during storage.
Ex: If the food is packaged in a bag with a little bit of air(e.g. ground meat),the aerobes will grow first and utilize O2. The environment will become anaerobic, in which anaerobes grow favorably.
Ex: In most food fermentations metabiotic growth is observed.
In Sauerkraut fermentation,4 different bacterial species grow in succession, one creating the favorable conditions for the next one.
First ,coliform grow produce acid and activate the growth of lactic acid bacteria.
second, Leuconoctoc mesenteroides ;
third Lb. plantarum
Last, acid tolerant Lb. brevis
Two or more microorganisms help one another during growth in food.
In yogurt;there are two types of lactic acid bacteria.
thermophilus produces small quantities of formic acid and stimulates Lb. Bulgaricus.
Lb. bulgaricus produce aminoacid inturn these products stimulate the growth Str. thermophilus
When two types of microorganism grow together and may able to bring changes which could not produce alone.
Acetaldehyde is desirable flavor component in yogurt.
thermophilus produce 8 ppm Acetaldehyde
Lb. bulgaricus produce 10 ppm Acetaldehyde in milk independently,when they grow together, they produce 30ppm Acetaldehyde .
Microorganisms may not effect each other but one organisms uses the substrate whic isproduced by other.
For ex: cellulose hydrolyzing microorganisms produce glucose and cellulose non hydrolyzing micoorganims use this glucose.
One population benefits while latter remain unaffected.
Microorganisms can adversely affect each other, one kill the other. Some Gr(+) bacteria produce antimicrobial components that can kill many other types.
For ex: L. lactis ssp. lactis produce bacteriocin called nisin and inhibits Gr(-)bacteria.
III. Chemical Changes Caused my microorganisms
1.Changes in nitrogenous organic compounds
2.Changes in organic carbon compounds
Characteristics of Predominant Microorganisms in Food
Prof Dr. Nihat AKIN
Selcuk University, Food Engineering Department
Characteristics of Predominant.Microorganisms in Food
CLASSIFICATION OF MICROORGANISMS
MORPHOLOGY AND STRUCTURE OF MICROORGANISMS.IN FOODS
IMPORTANT MICROORGANISMS IN FOOD
Important Bacterial Genera
Gram-Negative Facultative Anaerobes
Gram-Positive, Endospore-Forming Rods
Gram-Negative, Endospore-Forming Rods
Gram-Positive, Nonsporulating Regular Rods
Gram-Positive, Nonsporeforming Irregular Rods
Important Mold Genera
Important Yeast Genera
IMPORTANT BACTERIAL GROUPS IN FOODS
Lactic Acid Bacteria
Acetic Acid Bacteria
Propionic Acid Bacteria
Butyric Acid Bacteria
1. List the general differences in the morphology of yeasts, molds, bacteria, and bacteriophages important in food.
2. List the differences in the chemical nature and function of cell wall structures between Gram-positive and Gram-negative bacteria. How does this help determine Gram characteristics of an unknown bacterial isolate?
3. List four species of molds and two species of yeasts most important in food.
Food Microbiology lecture notes Dr. Osman ERKMEN
Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with about 1,500 species currently described.
• What are differences between yeasts and molds?
• What are differences between yeasts and bacteria?
• Pseudomycellium or pseudohyphae?
• “Pseudohyphae” are distinguished from true hyphae by their method of growth, relative frailty and lack of cytoplasmic connection between the cells.
• Yeast can form pseudohyphae. They are the result of a sort of incomplete budding where the cells remain attached after division.
• True yeast?
• Wild yeast?
• The cellular morphology of the yeast can vary from ovoid to long “sausage” shaped cells.
• The yeast produces large amounts of acetic acid when grown on glucose rich media (Asidogenic).
• They multiply by budding.
• They can use sugars as oxidative or fermentative.
• The most important species is B. intermedius.
– It can grow at as low as 1,8 pH.
– It can cause spoilage of beer, wine, non-alcoholic beverages and pickles.
• Candida genus was first described in 1923.
• Cells do not contain carotenoid pigment.
• Candida means “clear and bright white”
• Some species of this genus play role in fermentation of kefir, cacao, beer, ale and fruit juices.
• Some Candida spp. are commonly found in ground meat and poultry meats.
• While usually living as commensals, some Candida species have the potential to cause disease (aphthae).
• Many of them form films and can spoil foods high in acid and salt.
• Lypolytic Candida lypolytica can spoil butter and oleomargarine.