A HYGIENE DETECTION DEVICE

Technical Field

The present invention relates to a hygiene detection device which can be used in the fields such as food, water, agriculture, medicine, biology and chemistry, and that enables determination of toxic characteristics caused by the contamination of bacteria that has the ability to form indole by decomposing tryptophan amino acid.

State of the Art Presently, hygienic conditions need to be followed so that the human health is not jeopardized in fields such as food, water and agriculture. The microbiological values that need to be observed by the food products, arranged for the Enterobacteriaceae and E. Coli, according to the Microbiological Criteria of the Turkish Food Codex, is given in Table 1.

(1) n: Number of samples c: Number of samples allowed to have a value between the limits m and M

(2) Most Probable Number (MPN) management

(3) The analysis sample must be prepared from at least 10 different samples. (4) Unless indicated otherwise, considered as cfu/g-mL. Cfu: Colony-Forming

Unit (in agar media) Table 1 - Microbiological values need to be observed by food products

Presently, in order to detect the formed bacteria in fields such as food, water and agriculture, numerous different methods are tried. In most of these methods, and especially the traditional methods, first food or water samples are taken, and these samples are then enriched. This enrichment process takes 24-48 hours in average. Then, the cultures are planted by such methods as scratching or swabbing, and they are left for incubation for 24 hours. The reproduced bacteria are seen in petri dishes in different colors. Such a detection process for bacteria requires laboratory conditions, it is troublesome, exhausting and it takes too much time.

Apart from the traditional methods, the methods such as PCR, ELISA, Membrane Filtration, MUG Analysis Method are rather expensive and exhausting methods which also take too much time and require laboratory conditions.

The methods allowing detection under 24 hours are quick-detection methods. There are quick detection devices and quick detection sets for allowing this. The detection times have been lowered by these quick detection sets, however for the bacteria enrichment process before the detection, 18-24 hours are needed.

Table 2 and Table 3 discloses the total number of samples arriving at the 99 private food control laboratories as of 2017, according to the annual report of 2017 of the Directorate General of Control of Ministry of Food and Livestock of

Turkish Republic. Table 2 - Number of samples of food control laboratories affiliated with the Ministry of Food and Livestock of Turkey as of 2017

Table 3 - Number of samples of food control laboratories authorized by the Ministry of Food and Livestock of Turkey as of 2017

There are a total of 776,326 samples in these two tables. Total number of food and feed samples is 738,857, number of water samples is 37,469.

The price list of the Ankara Food Control Laboratory Administration as of 2017 for the analysis of Coliform Bacteria / Number of fecal bacteria in food and feed for 1 kg is 171 TL and analysis of Coliform Bacteria / Number of fecal bacteria in water for 1 It is 155 TL.

When taken into account that the food samples are 250 g in average and the water samples are 250 ml in average, it can be seen that there are 184,714.25 kgs of food and feed, and 9,367.25 kgs of water samples. Meaning that a total of 33,038,060.5 TL is spent for Coliform bacteria / Fecal bacteria analysis, 31,586,136.75 in average being for food and feed, 1,451,923.75 in average being for water.

Numerous patent applications are present in the state of the art for determination of bacteria. In the patent application numbered JP2000270894A, determination of E. coli using derivative of D-luciferin-O-Beta-D-glucuronide is disclosed. In the patent application numbered WO1998048046A2, determination of pathologic E. Coli in a sample wherein the DNA isolated from the said sample using oligonucleotide primers specific for E. Coli is amplified by PCR is disclosed. In the patent application numbered W02004087942A2, contacting a subject with a detectably labeled substrate for an enzyme produced and/or secreted by Escherichia coli is disclosed. In the patent application numbered US20070196884A1, usage of an inducing solution for quickly detecting the coliforms which can induce the b-glucuronidase and b-galactosidase expressions of the inducible enzymes in absence of cell growth is disclosed. In the patent application numbered CN103822949A, detection of Escherichia coli using an electrochemical biosensor is disclosed. In the patent application numbered US6060252A, primer target binding sequences, amplification and detection of Shigella and EIEC targets in various amplification and detection reactions are disclosed. In the patent application numbered JP5826005B2, determination of E. coli using preparation of E. coli detection media is disclosed.

In conclusion; because of the above-mentioned problems, and due to the insufficiency of the present solutions, a development is needed in the related technical field.

Objects of the Invention

The present invention is developed by the inspiration of the state of the art, and it aims to solve the above-mentioned problems.

The objective of the invention is to determine the toxic characteristics caused by the contamination of bacteria that has the ability to form indole by decomposing tryptophan amino acid, which can be used in the fields such as food, water, agriculture, medicine, biology and chemistry.

Another objective of the invention is to provide insight about the consumability of foods. Yet another objective of the invention is to reveal the contamination of ready-to- eat foods.

Yet another objective of the invention is to detect the products having low hygienic qualities by search tests.

Yet another object of the invention is to reduce the workload of the food control laboratories.

Yet another object of the invention is to provide added value as per community health. Yet another objective of the invention is to provide a solution that is quick, economic and portable.

The structural and characteristic features of the invention, and its advantages, will become apparent by the virtue of the drawings, and the detailed description with reference to the said drawings, and for this reason, the said drawings and the detailed description should be taken into consideration during review.

Drawings

Figure 1 is a schematic illustration of the inventive hygiene detection device. Reference numerals 1. Hygiene detection device

2. Battery

3. Test cartridge

4. Resistor

5. Thermometer setup 6. Optic Reader

7. Control unit

8. Screen

Detailed Description of the Invention

In this detailed description, the preferred embodiments of the inventive hygiene detection device (1) are disclosed for a better understanding of the subject matter.

The present invention relates to a hygiene detection device (1) which can be used in the fields such as food, water, agriculture, medicine, biology and chemistry, and that enables determination toxic characteristics caused by the contamination of bacteria that has the ability to form indole by decomposing tryptophan amino acid.

The proposed hygiene detection device (1) enables detection of toxic characteristics caused by the contamination of bacteria that has the ability to form indole by decomposing tryptophan amino acid. E. coli and V. cholera can secrete indole up to 0.6 mM in rich media. By the virtue of the secreted indole, discrimination between the types of bacteria (Salmonella (-), Edwardsiella (+), E. coli(+), Klebsiella (-), Enterobacter (+), and species (P. multocidea (+), P. haemolytica (-), P. mirabilis (-), P. vulgaris (+)) could easily be done by the inventive hygiene detection device (1). Especially the bacteria Escherichia coli, Klebsiella oxytoca ve Citrobacter, Staphylococcus aureus, Edwardsiella spp. are used as important indicators of the health quality of water and food, since they are present in intestinal flora. The inventive hygiene detection device (1) provides insight about the consumability of foods, reveals the present contamination of ready-to-consume foods, and detects the products having low hygienic qualities by search tests. By the virtue of the hygiene detection device (1) the workload of the food control laboratories is reduced and a quick, economic and portable solution which provides an added value in terms of the community health is presented.

Since the hygiene detection device (1) is used for detection of hygiene of food and water, it prevents the spreadable diseases and the food-related deaths. Renal failures, diarrhea, vomiting and the economic turmoil and social traumas caused by insufficient hygiene are reduced by means of the hygiene detection device (1). By the use of the hygiene detection device (1), the community health is improved and the length of the treatment process of the patients and the economic expenses during the treatment are reduced. The hygiene detection device (1) could be used for determining the contamination level of the waters and is therefore efficient in management of water resources. For this reason, the commercialization potential of the hygiene detection device (1) in the fields of food, water, agriculture and especially in health sector, and at the same time, it has a wide field of application. The hygiene detection device (1), as indicated, provides a study about the bacteria that has the ability to form indole. Indole is a nucleus detecting molecule which mediates the intercellular signals in bacteria. Indole has various important applications in pharmaceutical industry and is a bioindicator in biological and clinical samples. Specific analysis for detecting the indole amount are compicated and they require expensive equipment and high-level education. For this reason, Kovak’s test, which non-specifically detects indole analogues in samples, is used. For example, in the presence of 5 mM of tryptophan, E. coli bacteria could produce about 3.3 mM of indole. The indole controls various aspects of bacterial physiology in indole-producing bacteria such as spore formation, plasmid stability, drug resistance, biofilm generation and virulence. Some of the Gram positive bacteria of nearly 85 indole-producing bacteria species are as follows: Bacillus alvei, Clostridium novyi, Clostridium limosum, Clostridium tetani, Corynebacterium acnes, Desulfitobacterium hafniense, Enterococcus faecalis, Symbiobacterium thermophilum. Some of the Gram-negative bacteria are as follows: Aeromonas hydrophila, Aeromonas sivilar, Bacteroides sp., Chromobacterium violaceum, Citrobacter freundii, Citrobacter koseri, Edwardsiella tarda, Enterobacter aerogenes, Escherichia albertii, Escherichia coli, Escherichia fergusonii, Flavobacteria, Fusobacterium polymorphum, Haemophilus influenzae, Klebsiella ornithinolytica, Klebsiella planticola, Pantoea topaklan, Pasturella pneumotropica, Proteus vulgaris, Pseudovibrio sp., Shigella boydii, Shigella flexneri, Sphaerophorus varius, Vibrio cholerae, Yersinia intermedia, Yersinia enterocolitica, Yersinia frederiksenii and Yersinia kristensenii.

In this study, the bacteria to be detected are the bacteria that has the ability to form indole using the tryptophan. These bacteria are indole positive bacteria. The most important ones of these bacteria, Edwadsiella, Klebsiella, Vibrio and especially Escherichia coli are indicators of fecal contamination. These bacteria have the ability to form indole by decomposing tryptophan, which is an amino acid. Indole is a test enabling to distinguish between the genus and species of bacteria, especially most coliform bacteria. Indole test is as follows: The inventive hygiene detection device (1) takes advantage of biological reactions during its utilization. The inventive hygiene detection device (1) has determined that the bacteria synthesize sufficient indole in 4-8 hours. The microorganisms are incubated at 37 °C for 24-58 hours after being passaged to a suitable broth (generally TB). The reason for keeping it at 37 °C is that it provides a suitable temperature condition for both bacterial reproduction and for the bacteria to synthesize indole. As the bacteria in the sample reach a certain number at the end of incubation (4-8 hours), sufficient indole is produced.

0.5 ml of Kovac's separator is dripped on the cultures inside tubes. The formation of a red ring on the top part of the tubes indicates a positive reaction (indole formation). A yellowish ring indicates that no indole is formed (negative indole test result). As a result, it is determined if the sample is a bacterium capable of forming indole or not, and the genus and species of the bacteria could be distinguished. For example, while Klebsiella oxytoca is indole positive, most Klebsiella species are indole negative, or while Pasteurella multocida species is indole positive, Pateurella haemolytica species is indole negative. The change of color is read and shown to the user as indole positive or indole negative.

The detection of the bacteria such as E. coli and Klebsiella Pneumoniae which may be present in food and water, and which may cause toxic effects, is realized in a quick, economical, and reliable manner. At the same time, the required data could be reached using techniques without the requirement of laboratory conditions.

Considering the food industry, the hygiene detection device (1) has a major importance for testing the convenience of hygiene and sanitation conditions, during the harvest and transportation of food products in farms and ranches or during its processing in factories, during packaging, during its distribution at markets, restaurants and food serving places or homes. A quick and practical testing is provided based on the traceability of the hygiene of food, water and feed in this chain. Not being able to provide this safeness causes distrust and loss of customers, reduction in consumption and sales, loss of reputation and reduction in market share. More important from these economical losses is the harm done on the human beings by these non-hygienic conditions. The detection of the hygiene of food and water in short time will make it easier for the food products to be produced and served for consumption in suitable conditions and in conformity with the regulations. The hygiene detection device (1) could also be used in institutions such as medical institutions, food and water producing companies, hotels, cleaning and sanitation companies, restaurants, military units, recreational facilities, businesses selling raw meat, businesses producing ready- to-consume food, textile factories and such entities. The hygiene detection device (1) can detect fecal bacteria in analysis of drinking water and utility water and food, in a short time. The hygiene detection device (1) schematically illustrated in Figure 1 comprises;

❖ at least one battery (2) which provides the electrical power required by the components in order for them to work,

❖ at least one test cartridge (3) which amplifies the bacteria level from 10 ³ to 10 ⁵ such that the indole reaction takes place and the red ring occurs, ❖ at least one resistor (4) for heating the tube having the food or water sample passaged with a broth (generally TB) having tryptophan amino acid, and obtaining optimal temperature, thereby allowing the bacterial reproduction and preserve aliveness,

❖ at least one thermometer setup (5) enabling the incubation continuously for 4-8 hours at the temperature, 37 °C, obtained by the said resistor (4), enabling the enzyme produced by the bacteria in this incubation to react with tryptophan amino acid, and thereby enabling the production of sufficient indole from the tryptophan amino acid by way of deamination,

❖ at least one optical reader (6) for sensing the red color formed on the upper part of the tube upon the reaction of the indole produced by the bacteria with the Kovak's reactive, after adding on the tube a sufficient amount of the Kovak's reactive which is a biochemical consisting of isoamyl alcohol, paradimethylaminobenzaldehyde and hydrochloric acid, after the bacteria reaches a certain level after the incubation period and produces indole, if there are bacteria present in the food or water sample which are kept below a certain temperature,

❖ at least one control unit (7) which receives the temperature data from the said thermometer setup (5), which enables to hold the food or water sample at an optimum temperature by determining the operating manner of the said resistor (4) according to the said data, which analyses the frequency of the red color from the data it receives from the optical reader (6), which detects upon the said analysis that the said food or water sample contains a bacteria capable of forming indole or not, and which distinguishes the genus and species of a bacteria present in the said sample, and

❖ at least one screen (8) for showing the results of the analysis conducted by the said control unit (7) In classical enrichment processes, the number of bacteria is amplified from a level of 10 ³ to a level of 10 ⁸ . Using the test cartridge (3) of the invention, the number of bacteria is amplified from a level of 10 ³ to a level of 10 ⁵ . This amount is sufficient for the indole reaction to take place and for the red ring.

TB is a broth containing tryptophan. The tryptophanase enzyme produced by E. coli or such bacteria reacts with the tryptophan, and pyruvic acid, ammonia and indole is formed from the tryptophan by deamination.

In the inventive hygiene detection device (1), an incubation environment of 37 C is provided for the bacteria, and the bacteria is left for incubation at 37 C for 4-8 hours. Upon the warning received after 4-8 hours, 0.5 ml of Kovak's reactive is dripped on the indole that is produced by a bacteria such as E. coli in a sufficient amount, that is, it contains sufficient indole for detecting the maroon layer formed on upper surface of the Eppendorf in which the reaction takes place.

The Kovak's reactive is a biochemical reactive consisting of isoamyl alcohol, paradimethylaminobenzaldehyde and hydrochloric acid. The indole produced by E. coli and such bacteria reacts with the para-dimethylaminobenzaldehyde, and they form a maroon colored layer on the surface of the Eppendorf in which they are located. The optical reader (6) senses the frequency of this maroon color, and the presence of bacteria, if any, is shown to the user via the LCD screen (8).