Aflatoxins are extremely toxic fungal metabolites found in human and carnal nutrients. Aflatoxin B1 is the most carcinogenic and mutagenic metabolite nowadays in animate being, feedstuff and alterations to Aflatoxin M1 in milk. Aflatoxin M1 has a considerable hygienic hazard for human wellness. Previous researches have been shown different attacks for decontamination of this metabolite. Removal of Aflatoxin M1 by chemical, physical and biological methods has been studied. In old surveies, some species of Fungis and bacteriums have been demonstrated to degrade or adhere the Aflatoxin M1 in liquid media.
In this research, two species of Lactic Acid Bacteria ( LAB ) are used. Lactobacillus delbrueckii subsp. Bulgaricus and Streptococcus thermophilus have the of import function in yoghurt production and agitation. In this survey, adhering activities of these two strains will be assessed in milk and yogurt and binding degrees of strains will be compared with each other. Besides, the degrading ability in milk and yogurt will be considered and compared. Then the treatment will be made that which type of merchandise has been shown more binding ability and analyzing the chief factors and chief grounds that will be done for the achieved consequences.
Mycotoxins are the secondary toxic metabolite of Fungis, produced in the terminal of logarithmic growing stage. Production of mycotoxins depends on handiness of sufficient and suited of indispensable foods for growing, suited environment such as the specific humidness, temperature, PH, and the presence of toxigenic molds. They affect on the undermentioned factors: growing rate in immature animate beings and babies, interfere in mechanism of organic structure, fondness on immunological system and cause reactivity ( Pier, 1981 ) .
Higher degree of these factors leads to decease by damaging the kidney and liver ( Hayes, 1980 and Dvorackova, 1990 ) . Harmonizing to the Food and Agriculture Organization ( FAO ) ,25 % of the universe ‘s harvest contaminated by mycotoxins ( Giessen, 1998 ) . Aflatoxins are one of the chief groups of mycotoxins. They have extremely toxic, mutagenic, carcinogenic, teratogenic and immunosuperssive effects on assorted carnal species and homo.
They are produced by three strains of fungi Aspergillus flavus, A.parasiticus and A.nominus. Aflatoxin ‘s best temperature for growing is between 12oc- 40oc ( Koehler et al. , 1985 ) . Four types of toxins have been found in these metabolites which are divided in two groups B ( B1, B2 ) and G ( G1, G2 ) . Aflatoxin B1 has the inveterate toxic effects on mammals produced by A.flavus and A.parasiticus ( Palmgreen and Hayes 1987 ) .
Aflatoxin B1 is normally found in carnal grocery and taint occur during harvest home, storage, processing, transporting and silage. Corn, peanuts, cottonseed and rice are the chief harvests contain the high degree of Aflatoxins. Aflatoxin M1 ( AFM1 ) is the hydorxylated metabolite of Aflatoxin B1 in the milk.
This metabolite was foremost detected in cow milk. It had been fed the contaminated maize and peanut by Aflatoxin B1. There is a relationship between the sum of AFM1 in the milk and ingestion of contaminated grocery by AFB1 ( Sassahara et al. , 2005 ) . After consumption and digestion of AFB1 by ruminant, it is synthesized to AFM1 in the milk. Metabolizing of AFM1 occur in animate being ‘s liver ( Frobish et al. , 1988. , Van Egmond and Paulsch, . 1986 ) . Previous studied have been estimated that about 0.3- 6.2 % of AFB1 in animate being ‘s ration convert to AFM1 in milk ( Creppy, 2002 ) . AFB1 transition to AFM1 includes four stairss. The carcinogenic authority and toxicity of aflatoxin M1 is 10 times less than aflatoxin B1 but the acutely hepatotoxic is same as AFB1 ( Cullen et al. , 1987 and Lafont et al. , 1989 ) . The European Commission ( EC ) has accepted a bound of 50 ng/l for AFM1 for milk ( European Commission, 2001 ) while the US nutrient and Drug Administration ( US FDA, 1996 ) has recommended the maximal degree of 500 ng/l for AFM1 in milk.
In existent fact, taint of milk and milk merchandises by AFM1 depends on several environmental factors such as season, geographics, state. The degree of AFM1 in dairy merchandises is variable between hot and cold conditions. In spring and summer, animate beings are fed by grass, grazing land, weed while in winter dressed ores used less than leafy vegetables. It causes a autumn in AFM1 degree ( Galvano et al. , 1996 ; Pitet, 1998 ; Sarimehmetoglu et al. , 2003 ) .
In old surveies, AFM1 is non destroyed during the heat processes like pasteurization or alterations are non obvious ( Yousef and Marth, . 1989 ) . Besides, in yogurt and cheese production the pollution degree of AFM1 does non alter and remains stable. ( BARBIERI et al, 1994 ; CREPPY, 2002 ; GALVANO et al. , 1996 ; PITET, 1998 ) . Another survey has been confirmed 9 % of addition in AFM1 when the yogurt was produced from contaminated milk ( Van Egmond et al. , 1977 ) .
The best manner to avoid the presence of AFM1 in dairy merchandises is to command the carnal grocery during crop, storage and maintain them in suited status to forestall AFB1 formation. Different methods and factors are available and have been tested before to detoxicate the aflatoxins from nutrient and provender stuffs. Three methods are common to utilize to degrade the degree of aflatoxins in nutrients and provenders. Chemical, mechanical and biological methods are applied to diminish the degree of taint in different types of nutrients. The FAO needs some conditions to accept the decontamination procedure. Any detoxification process must extinguish, demobilize or destruct toxins. Decontamination method should non bring forth any toxic in the concluding merchandises. These methods must take any fungous spores that could increase and bring forth new toxins.
In physical detoxification, heat interventions, gumma radiation, UV or seeable visible radiation are applied. Chemical debasement is more practical and reasonable method. Sodium bisulphate
Converts AFM1 to H2O soluble merchandise ( Doyle and Marth, . 1978, Hagler et al. , 1982 ) . Treatment with ammonium hydroxide, Ca hydrated oxide, methanal and clays are the chemical methods to cut down the hazard of aflatoxicosis. Some strains of lactic acid bacteriums ( LAB ) and barms have been reported to degrade of aflatoxins level in dairy merchandise ( El- Nezami et Al. , 1998 a ) .
In this research, the biological method will be applied to adhere the ability of AFM1 in milk and yoghurt. Two types of Lactic Acid Bacteria ( LAC ) , Lactobacillus delbrueckii subsp. Bulgaricus and Streptococcus thermophilus, will be used for this intent.
Lactobacillus delbrueckii subsp. Bulgaricus is used for yoghurt production and found in other fermented merchandises. This strain is regarded as acidophilous bacteria PH ( 4.6- 5.4 ) and unable to ferment any sugar except Lactose. During fermenting to yogurt, it produces ethanal that is one of the yoghurt olfactory property elements. Streptococcus thermophilus converts lactose to lactic acid and ethanal. It is counted as a probiotic. In add-on, this strain alleviates the symptoms of lactose intolerance.
In this method, samples of assorted types of milk and yoghurt will be collected from different stores. Whole milk, semi-skimmed milk, skimmed milk, low fat yogurt and high fat yogurt are traveling to be used in this research.
The research processes include, civilization readying, taint with AFM1 in PBS, milk taint with AFM1, yogurt taint with AFM1, statistical analysis and consequences.
At first, Lactobacillus delbrueckii subsp.Bulgaricus and Streptococcus thermophilus is cultivated and incubated so these bacteriums are centrifuged and washed with buffer. The following measure is suspending bacterial pellets in PBS, which is contaminated with AFM1. Then the bacterial pellets is incubated and centrifuged. Extracting the unbound AFM1 will be done by Immunoaffinity Columns ( IAC ) method and the process will be completed harmonizing to R-biopharm GmbH recommendation. This method will be repeated in milk and yogurt samples. Again, prepared bacterial pellets will be suspended in contaminated milk or yogurt with AFM1. Removing the unbound AFM1 will be done by IAC method after the incubation and extractor of milk and yoghurt samples.
The statistical analysis will be based on the discrepancy comparing. Differences between discrepancies show the difference between adhering sums of AFM1 in two medium of two bacteriums. ANOVA and DUNCAN trial are suggested analyses methods in this research.
The most of import thing in this undertaking is showing the adhering ability of LAB. Which type of these two strains has a more binding ability in comparing of another one? In add-on, another mark of this undertaking is to place the binding ability of milk and yogurt. Which merchandises adhering capableness is much greater than other? Alternatively, does the sum of fat have any consequence on AFM1 debasement? All the consequences will be discussed. The biological and chemical environment of each merchandise must be considered. To happen out the chief ground several factors must be intended. For illustration, medium PH, available enzymes in the merchandise, agitation, types of protein in each merchandise, sum of fat and type of bacteriums. To accomplish the best determination, utilizing the old researches related to this topic is advised. After accurate probes and sing all facets of this research, the concluding decision will be suggested.
Different methods are available to pull out the unbound AFM1 from supernatant. Thin Layer Chromatography ( TLC ) , High Performance Liquid Chromatography ( HPLC ) , Enzyme Linked Immunosorbent Assay ( ELISA ) and Immunoaffinity Columns ( IAC ) . Immunoaffinity column is the most suited method for this research. This method purifies the substance from a mixture into a buffering solution, cut down the sum of substance in a mixture and purify and concentrate the enzyme solution.
The end of this undertaking is to diminish the degree of AFM1 in dairy merchandises with biological method to avoid the harmful consequence of this aflatoxin and suggest the new technique for this planetary job.
The chief aims include:
O Roll uping the samples
O Fixing the LAB and AFM1
O Adding the LAB and AFM1 to supply samples
O Detecting the unbound AFM1 with IAC method
O Statistical analysis by ANOVA and DUNCAN trial
O Geting the consequences
To avoid prejudice in this undertaking some facets must be considered
O Choosing the appropriate statistical trial
O Sing the immaterial factors
O Accuracy in sample collection
O Applying sufficient volume and degree of used stuffs
O Sing the truth of used equipments
Submit Grant Proposal
Expected Grant Notification
Obtain Materials and Equipments
Project Start up
Prepare Results Report
Aflatoxin M1 has been related with assorted diseases. It is diagnosed farm animal, domestic animate beings and homo in the worldwide. Certain environmental factors cause the happening of aflatoxins in animate beings. Such as geographical location, agricultural and agronomic process, bring forthing of Fungis that cause mycotoxins presence during preharvest, storage and processing.
Aflatoxin M1 cause liver and kidney disfunction and malignant neoplastic disease in animate being and human. These are well depends on age, sex, species and nutrition. Clinical symptoms in carnal include ammonium hydroxide, reduced reproductivity, disfunction in nutrient use and GI jobs
Evidence of aflatoxins wellness impact on homo has been reported from different country of universe particularly in 3rd universe states and the acute status of disease leads to human decease.
Overall, because of all the issues that mentioned above aflatoxins have the irreparable impact on the universe economic system. Ineluctable presence of aflatoxins in nutrients and provenders and do the harmful consequence on homo and animate beings leads to detect and research the new methods for detoxification and degrading of these mycotoxins.
In this undertaking, it is attempted to measure the consequence of lactic acid bacteriums on diminishing the degree of AFM1 in milk and yoghurt. If the result can be acceptable, it will be used for the remotion of AFM1 from dairy merchandises.
The other account to utilize LAB for decontamination of AFM1 is the side effects of some mechanical and chemical methods. These techniques are non appropriate to use in dairy merchandises and the biological method in these instances has been advised
SOLID AFM1 ( SIGMA )
Lactobacillus delbrueckii subsp. bulgaricus
Phosphate Buffer Saline ( PBS )
Milk & A ; Yoghurt
Pipets 1-10 milliliters
9 UV Light box and beginning ?500
10 Other equipments ?300
11 TOTAL ?5350
AFLASCAN KIT COMPONENTS
Aflascan unsusceptibility columns
Glass syring barrel
Plastic syring pump unit
Fluroscent comparator card