Fusarium

Fusarium is other types of toxigenic molds that associated with the cereal grains, oil seeds, corn, wheat, barley and the grains products. In the finished flour, F. graminearum and F. verticillioides (moniliforme) are the common Fusarium species that can be found in corn. Thus, the major toxins that link to human health are deoxynivalenol, zearalenone and perhaps fumonisins. The types of mold that can contaminants the food are shown in the following table:

Detection, Isolation and Identification of Fusarium Species

Isolation of the Fusarium Species is done using selective media in which either the direct plating (placed the grains in the media) or by plate count serial dilution of the food.

Media use to isolate and detect Fusarium species

A. Nash-Snyder Medium } use to evaluate sample that has high counts with
B. Modified Czapek Dox (MCZ) agar } bacteria or fungi

C. Czapek iprodione-dichloran (CZID) } Not for rapid identification unless subcultured in CLA (carnation leaf agar)

D. Potato-dextrose- iprodione-dichloran (PDID) } support Fusarium with morphological
and cultural characteristics

E. Dichloran- chloramphenicol-peptone agar (DCPA) }inferior than the CZID and PDID

The Macronidia and Microconidia is accessed for the identification in term of their morphology and structures.

Detection of the Fusarium Toxins

1. Sampling: to ensure a sample used is representative of the whole load
2. Extraction: the sample is dissolve in organic solvent ( methanol, acetone, acetonitrile )with
some water.
3. Filtration and clean up: to remove any residue and interfering substance. Cleanup can be done
by passing the extract through sorbent packed column.
4. Concentration: can be done by mild heating
5. Separation of components: thin-layer chromatography (TLC), High-performance liquid chromatography (HPLC) or Gas-chromatography (GC).
6. Quantification and Identification: TLC is used to test Deoxynivalenol. Or HPLC with 219nm
UV absorbance. Deoxynivalenol used HPLC with fluorescence detection. Whereas for fumonisins, TLC is employed for screening.


Besides the above test, commercial ELISA kits are also available. Kits for qualitative and quantitative of Deoxynivalenol and Deoxynivalenol are available. In addition, antibody-based affinity column for fumonisins.

References

Michael P.Doyle and Larry R. Beuchat. Food Microbiology: Fundamentals and Frontiers; 3rd Edition. ASM Press; United States of America. Accessed on 16 July 2008.

Aspergillus

Aspergillus species is commonly found in the corn. The species that is closely associated with the corn is the Asp.flavus and A.parasiticus. The mycotoxin formed by these 2 species is Aflatoxins.[1]

Aflatoxins is then classify B and G. In maize Aflatoxins B is present in it and this might remain in the flour as the maize under minute processing treatment when milled into flours.

Source of Aflatoxigenic mold and Aflatoxins
Aspergillus Flavus and parasiticus occur naturally. They can invade the crops before harvesting. In corn, the molds can enter when the crops are damage by insect or during the development of the ear of the corns. Both the species are usually found in peanuts, oilseed, cottonseed and corn. A. flavus can also be discovered in cereals and spices.

Detection and Identification of Aflatoxigenic molds
The medium used to detect the Aflatoxigenic molds is Aspergillus Flavus and parasiticus agar. This agar is specifically made to detect this toxin. The agar is then incubated at 30°C for 48 to 72 hours. The Asp.flavus and A.parasiticus then will produce an orange-yellow colony reverse in which it can be easily being recognized and detected.

Hazards of Aflatoxins
Aflatoxins can result in acute health hazards which include lung, liver cancer and teratogenesis. In addition, it will lower the human immune system and affect the production of the antibodies in the body. Thus, this result the bodies to be prone to infectious disease.

Detection of toxins
The samples are extracted with solution consisting of the organic solvent (methanol and chloroform) and small amount of water. The presence of food composition such as protein, fats or pigment will interfere the effectively of the extraction. In this cause, solvent such as the hexane can be employed to separate these interference from the sample.

The extracts are then undergoes clarification whereby the extracts are concentrated and cleaned up to remove or filter out any possible food particle residue. Lastly, the extract are then separated by thin-layer chromatography or HPLC. The Aflatoxins will be visible under the UV light and are quantified by fluorimetry or by the comparison with the known standard concentration, if thin-layer chromatography is being used.

Immunoassay techniques such as ELISA and dipstick tests can also be used for the detection. Whereas, rapid technologies like the use of biosensors, PCR can commercial test kits are also available for detecting the toxin.

References for above info and data:

1. ICMSF. 2005. Micro-organisms in Foods 6; 2nd Edition. Klumer Academic/ Plenum Publishers; USA. Accessed on 10 July 2008.

2. (mostly from)

Michael P.Doyle and Larry R. Beuchat. Food Microbiology: Fundamentals and Frontiers; 3rd Edition. ASM Press; United States of America. Accessed on 16 July 2008.

Detection and Identification

These 2 words normally used in conjunction in the rapid method. However, they mean two different things. Identification refers to determine the identity of the pure culture isolate whereas detection is the detecting of the microorganisms directly from the food.

In the past, the analysis of the microbiology involves 3 steps. The 1st step is the pre-enrichment step in which the food are incubated in non selective media for microbial cell repair. The 2nd step is the selective enrichment whereby the culture (pathogen of the interest) are then transferred to the selective media for growth while limiting the growth of undesirable pathogen. Lastly, the last step is the post enrichment. In this steps, the pathogen of the interest are isolate in to pure culture for growth in order for the identification method to take place. However, this conventional method is time-consuming and labor intensive. Therefore, rapid methods are created to speed up the identification and detection for effective analysis.

Identification techniques or methods

A. Miniaturized Biochemical and other identification Assay

The identification assays usually involve the use of pure isolate of unknown pathogen and it is mostly identify based on the biochemical characteristics of the micro-organisms. Identification based on the microbials’ biochemical characteristic is tedious and require time-consuming which may take weeks or months to analysis. The introductions of the miniaturized biochemical kits enable to analysis the biochemistry of the micro-organisms in a faster time. These kits contain 115 to 30 types of media or substrates and only require 18-24 hrs of incubation before the result can be obtained. Beside using the biochemistry for the accessing the type of organisms, test kits can also make use of the fatty acid or nucleic acid for the identification.


Corn flour may contain Salmonella and maybe B. cereus. Note: Salmonella spp. belong to Enterobacteriaceae family.


Antibody-Based Assays and Nucleic Acid-Based Assays can also used to detect and identify the micro-organisms and toxins.

The Nucleic Acid- Based Assays include the DNA probes, PCR and Bacteriophage. These assay is used to detect pathogen in the food. Examples of DNA probes are the GENE-TRAK asaay in which 2 types of method are applied- DNA hybridization and enzyme immunoassay. PCR involves hybridization and amplification. It is effective and fast, however, it can be easily distributed by the composition of the food. Lastly, Bacteriophage is used to detect specific pathogen in the foods. Examples of it are the bioluminescence and the ice nucleation.

The simplest test of the Antibody-Based Assay is the Latex Agglutination in which antibody-coated colored latex beads or colloidal gold particles are used to test the bacterial cell suspension. For the analysis of toxin, Reverse Passive Latex Agglutination is normally used.

There are many other Antibody-Based Assay test which include Immunodiffusion, Immunoprecipitation, ELISA (Enzyme-Linked Immunosorbent Assay) and Immunomagnetic Separation (IMS).


Some of the tests mentioned above are now currently being improved with new technologies and to able to give resulting in a much shorter time with high accuracy and sensitively

PCR has been improved to rtPCR (real-time PCR) in which the results can be obtained in real time, meaning the data can be collected immediately during the certain processing or analysis steps.
Moreover, Biosenors and DNA Microarrays are also developed. Biosensors make use of the minute fluctuations resulting from the biological interactions into measureable digital electronic reading. On the other hand, DNA Microarrays uses DNA chips, biochips , gene chips or genome chips, which capable of analysize thousand of gene simultaneously

Cereals

A. Fungi

The cereal mentioned here are the grains of wheat, maize, oats, rye…The fungi found in the cereals can be classified into Field fungi and Storage fungi.

Field fungi are those grow on the plants and invade the grains or seeds before the harvesting. The water activity for the Field fungi to grow are > 0.90 (= to 20-25% moisture). The damage done by the Field fungi includes blemishing, discolouration, loss of germinability and mycotoxin production, which occurred before harvesting or during the drying. However, the field fungi cannot growth during storage; they might slowly die during the storage period or that remain viable for a long period. Note: there are still some field fungi that are able to survive in low water activity.

Storage Fungi degrade the quality of the grains after the harvesting. Storage fungi are able to strive at low water activity.

There are 2 types of Field fungi which is commonly present in the maize- Fusarium and Aspergillus. Flavus. The common Fusarium fungi that is present in the Fusarium verticillioides. Other species include Fus. graminearum and Fus. subglutinans. The optimum growing temperature is > 30°C. The maize crops in the Asia are more prone to Asp. flavus than the crops in cooler area such as Midwestern US. The maize can also pre-harvest infected by the Penicillium species. These species include Pen. oxalicum, Pen. funiculosum and Pen. citrinum.


B. Bacteria

Bacillus, Lactobacillus, Pseudomonas, Streptococcus, Achromobacter, Flavobacterium, Micrococcus and Alcaligenus are bacteria that can be found in the cereals. Spore forming bacilli are the main bacteria in the cereal as they can survive in heating and strive if the water activity is not well controlled. Below are the ranges of each bacterium that can be present in the cereal:

C. Factors that effect the growth of microorganisms

Water activity: the water activity is critical in preventing the growth of storage fungi as they are capable of growing at low water activity.
Eurotium, which is an ascomycete state of Aspergillus are the major storage fungi found in grains. Below shows the minimum Aw and the approximate moisture contents of the storage fungi:

Temperature: The optimum temperature for most fungi grow is 10-35°C. In tropical region, Aspergillus are the cause of grain spoilage in which the optima temperature is 30-40°C, whereas the Penicillium is major cause of spoilage in the temperate climate whereby the optimum growth temperature is 20-30°C.


D. Presence of Toxin (mainly focus on maize)

Aflatoxins: It is produce by the Asp. Flavus. The Aflatoxins that usually present in the maize is B Aflatoxins.

Fumonisins: It is form the Fusarium verticillioides which produce toxin Fumonisin B. It may result in the human esophageal cancer.




Reference ( All info and tables are obtained from the below references):

ICMSF. 2005. Micro-organisms in Foods 6; 2nd Edition. Klumer Academic/ Plenum Publishers; USA. Accessed on 10 July 2008.

Flours

Flours are formed from the milling and tempering of the grains ( rice, maize, wheats etc). Thus, it is important to ensure that the initial microbial counts of the grains are within the acceptable range. In addition, the cleanliness of the processing equipments can also help in reducing the microbial load in the flour. Any residue left behind in the equipments will contaminate the flour.

Flour with moisture level less than 12% will not support any microbial growth. Moisture from the atmosphere, equipment or condensate can result in microbial growth in flour.

The fungi in the finished flour are usually Penicillium, Eurotium species and Asp. Candidus.

Spoilage of flour

If the moisture level of the flour is above 12%, xerophilic fungi will grow and that when reaches 17% result in bacteria grow. The growth of bacteria is faster than the fungi, thus it will become the dominant pathogen in flour when the moisture level has exceeded the limit. In the “exposed” flour, the lactic acid bacteria will cause acid fermentation, followed by the yeast’s alcoholic fermentations and lastly the oxidation to form acetic acid by the Acetobacter. This is uncommon in stored flour. Even without the presence of the lactic acid bacteria, micrococci and Bacillus spp. will also produce lactic acid., alcoholic and other phenolic compounds.

Pathogen and toxin production

Dry flour has a low water activities, thus does not support fungal growth in which prevent the occurrence of mycotoxin production.

Salmonella, is also a hazard in flours especially in flours that does not undergoes any heating treatment. It is because heating can kill or inhibit the Salmonella as they are heat sensitive.




Reference:

ICMSF. 2005. Micro-organisms in Foods 6; 2nd Edition. Klumer Academic/ Plenum Publishers; USA. Accessed on 10 July 2008.

Detection Methods of GMOs


1. The importance of having detection methods

Laws and regulations on the labeling of genetic modified foods have been established by countries. In order to establish these regulations, detection methods are needed as it enables the government sectors to detect and quantify the GMOs in foods. In addition, it provides transparency to the public or consumers on the GMOs in the food.[1]

2. Types of detection methods

Most of analytical tests on the GMOs are targeting on the DNA and protein [1]. The commonly use DNA-based method for the GMOs detection is the Polymerase Chain Reaction (PCR).

PCR involves 4 steps

(1): Sampling and sample preparation
(2): DNA purification/ Isolation of DNA
(3): PCR amplification and detection of reaction products
(4): Interpretation of results [1]

The first step of the PCR is the Sampling and Sample preparation. The sample use for the analysis is the representative of the whole lot of crops or products. Thus it is critical to ensure that the sample is not contaminated especially during the transportation [2]. Moreover, the sample size, particle size and the distribution of the raw materials also need to take into consideration as these factors will determine the number of modified gene being detected and the accuracy of the test [1] [2]. The economic of the sampling test are also one of the factors to be concern about.

All samples must be homogenized before conducting the test. Those crops sample such as soya bean, maize for to be incubated in sterile water for up to 20 hours and be blended for homogenization. Dry samples such as soya flakes or flour are to be incubated in sterile water and be homogenized, whereas, liquid samples must be well shaken before use [2].

The second step is the DNA purification. The purification process is to remove any PCR inhibitors that will affect the amplification of DNA in order to achieve a more reliable and accurate results. Examples of the PCR inhibitors are lipids, polysaccharides and polyphenols. There are 3 types of isolation procedures: the cetyl-trimethylammonium Bromide[1] (CTAB) (other references uses hexadecyltri-methylammoniumbromide[2]), DNA-binding silica columns and the combination of these 2 methods. Besides ensuring the purify of the DNA, the quality of the DNA is also important for the PCR process. The quality of the DNA is determined by the length of the DNA and the degree of degradation due to certain processing treatment.

The main step in the GMOs detection is the PCR amplification. PCR amplification enables a specific DNA target sequence to be amplified or multiple a billion times. It requires the help of the Primers, Taq polymerase and some heat treatment [1]. After the amplification, PCR-based GMO detections methods are then carried out. The methods can be classified into qualitative method or quantitative methods. Qualitative PCR amplification methods are used to determine whether any modified material are presence whereas the quantitative methods are used to find out the number (quantity) of modified material present in the foods. Quantitative methods are the commonly used methods as it is more precise and that it can specifically identify modified materials present. For instance, Event-Specific GMO detection methods belong to the quantitative methods [1]. Lastly, the interpretations of the results obtained from the above steps are carried out.

For the protein-based GMOs test, immunological methods are used. Examples of the methods are the enzyme-linked immunosorbent assay (ELISA) and the lateral flow test formats [1]. In terms of analysis time and the amount of sample needed, protein-based GMOs test are more quicker and the amount of sample needed are minimal with compare to the DNA-based GMOs test [1]. However, when in terms of the reliability and sensitively, DNA-based GMOs tests are more precise and accurate [2].

References:
1. Farid E. Ahmed. 2004. Testing of Genetically Modified Organisms in Food. 2004. The Haworth Press; United States of America. Accessed on 15 June 2008.
2. Knut J.Heller. 2003. Genetically Engineered Food; Methods and Detection. Wiley-Vch; Germany. Accessed on 15 June 2008.

Heinz sauce production

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