FIELD OF INVENTION
[001] The present disclosure relates to a dipstick for quick estimation of glucose in potato tubers. The present disclosure particularly provides a dipstick for quick estimation of glucose in potato tubers. The disclosure also provides a method for estimating glucose in potato tubers 5
BACKGROUND OF THE INVENTION
[002] Sugar accumulation in potatoes (Solanum tuberosum L.) is one of the most serious problems for the processing industry. Normally, potato tubers are stored at low temperatures (2-4°C) to control sprouting. However, the low temperature results in 10 accumulation of sugars. The accumulation of reducing sugars (primarily glucose and fructose) in cold-stored potato tubers is referred to as cold-induced sweetening. In potatoes, controlling the cold-induced sweetening is very difficult because the sugar content is affected by starch synthesis, starch breakdown, glycolysis and mitochondrial respiration, as well as hexogenesis (Davies et al. (1998), Pollock and ap Rees (1975); 15 Sowokinos (2001).Potato tubers contain reducing sugars (glucose and fructose) and (particularly glucose) participate in ‘Malliard reaction’ during making of potato products through frying, finally producing the unpalatable and dark colored finished product.
[003] The potatoes for seed purpose are stored at temperature of 2-4°C and is 20 beneficial for long term storage as it reduces the bacterial soft rots, decreases dry matter loss and prevents sprouting without application of any sprout inhibitors.
[004] However, the low temperature storage, leads to accumulation of glucose and fructose at substantial levels. As the respiration rate and sprouting are inhibited by low temperatures, the consumption of reducing sugars is low and their content accumulates 25 in the tubers, resulting in dark colored chips.
3
[005] In general, sugars tend to accumulate when their production is more as compared to utilization. Storage temperatures lower than 6ºC increases the activity of the enzymes that convert starch into fructose and glucose.
[006] However, potatoes stored at low temperature results in cold-induced sweetening. In other words the low temperature such as 2-4oC, is suitable for storage 5 of seed potatoes but it is not at all suitable for storing the potatoes meant for table and processing due to excessive accumulation of reducing sugars.
[007] For desired, processing quality of potato tubers, low reducing sugar are required and for their storage, temperature above 10oC is desirable but at this temperature sprouting needs to be controlled. Therefore, potatoes are stored at elevated 10 temperature (12±0.5°C) with CIPC treatment (Isopropyl N-(3-Chlorophenyl) Carbamate) for effective control of sprouting.
[008] For processing purpose, the farmers store their potatoes at 12±0.5°C with CIPC treatment for more than >6 months and send processing varieties to industry from time to time. Besides storage temperature, physiological age of the tuber also affects sugar 15 accumulation. Depending upon the condition of potato stored, bruised potatoes or with high sucrose content potato tubers at the time of storage sugar accumulation is more (later on converting to reducing sugars).
[009] Therefore, the suitability of potato tubers for development of processed products such as chips and French fries etc.is mainly determined by the amount of 20 glucose present in potato tubers. In general, glucose levels below 0.1 percent (i.e. 1000ppm) on a fresh weight basis are acceptable for chips and French fries production. In potato, the level of glucose is not static and changes do occur during the period between its harvesting and storage. The surrounding conditions mainly temperature has a great impact on the glucose levels accumulation in the potatoes. Storing the 25 potato tubers at low temperatures (4ºC or below) results in higher amounts of glucose in the potato tubers. As per the normal practice in India, farmers transport their potatoes to the processing industry and upon reaching the industries sites, their potato
4
consignments are rejected due to its higher glucose content by the industry after testing at their locations. This put the farmers in a difficult situation and leads to wastage of time and monetary losses in terms of transportation cost and rejection of the tubers. Thus, a simple and quick method for quick estimating the glucose level by the farmers and small processing units is needed. This helps both the farmers and small processing 5 units, which will empower them in taking appropriate decision for its post-harvest marketing /selling targets/ destinations.
[010] Benedict’s reagent is one of the possible technique to measure reducing sugars by mixing it with potato juice (0.5ml potato juice + Benedict reagent). The colour change is achieved by heating the content and observing the colour change. This 10 method has a limitation that the farmer is need to carry liquid reagent along with spirit lamp and match box, test-tubes, test-tubes holder, test-tube stand, one pipette measuring 0.5ml of potato juice and Benedict’s reagent etc.
[011] Another method could be using strips generally used to monitor blood glucose levels. The drawback is the cost of such strips and suitability of such strips. The blood 15 monitoring glucose strips are costlier.
[012] There are published literatures related to monitoring of body fluid glucose levels but these literatures are not related to estimation of reducing sugars in potato tubers.
References: 20
Costa, M.N., Veigas, B., Jacob, J.M., Santos, D.S., Gomes, J., Baptista, P.V., Martins, R., Inacio, J and Fortunato, E. (Nanotechnology, 2014, 25:1-12) discloses paper based microfluidics for molecular diagnostic testing.
Morin, L.G. and Prox J. (Clin.Chem. 1973, 19:9, 959-962) discloses use enzymatic procedure for determining glucose in serum or plasma by changing the assay at pH of 25 5.5.
5
Dubowski K. M. (Clin. Chem. 1962, 8: 3, 215-235) disclose use of chemical compound to obtain colour change.
In Lewis, G.G., DiTucci, M.J., Baker, M.S and Phillips, S.T. (The Royal Society of Chemistry, 2012), chromagen was used.
Davies, H.V. et al. (1998) disclose about prospects for manipulating carbohydrate 5 metabolism in potato tubers. (In proceedings of AAB Conf. on Protection and Production of Sugar Beet and Potato. Aspects of Applied Biology (52) (Dale, M.F.B. et al., eds), pp. 245–255).
Pollock, C. and ap Rees, T. (1975) explains activities of enzymes of sugar metabolism in cold-stored tubers of Solanum tuberosum (Phytochemistry 14, 613–617). 10
Sowokinos, J.R. (2001) disclose about biochemical and molecular control of cold-induced sweetening in potatoes (Am. J. Potato Res. 78, 221–236).
[013] In spite of the above published literatures, there is no simple method available especially for estimation of glucose in potato tubers in the fields or storage facilities by a farmer or field staff. 15
[014] Already known methods uses alkaline copper sulphate solution and such methods are cumbersome since, these methods require accessories like test-tubes, liquid reagents, burners, etc. The diabetic diagnosis strips primarily meant for glucose estimation in blood serum and reported for the estimation of glucose in potato may lack required sensitivity. 20
[015] The present disclosure thus pertains to development of one such simple and quick method for estimating the glucose levels in potato tubers.
OBJECTS OF THE INVENTION
[016] One of the objects of the disclosure is to provide dipsticks for quick estimation of glucose in potato tuber. 25
6
[017] Another object of the disclosure is to provide a simple and quick method for glucose estimation in potato tubers which obviates the drawbacks of the hitherto known prior art as detailed above.
[018] Another object of the present disclosure is to develop the media to dissolve the chromagens. 5
[019] Another object of the present disclosure is to identify the base material for development of reaction and to get the visual colour change with the naked eye.
[020] Still another object of the present disclosure is to identify the temperature for immobilization of chromagens.
[021] Still another object of the present disclosure is to develop a method for 10 preparation of potato samples for estimation of glucose.
[022] Another object of the present disclosure is to study the shelf life of the developed dipsticks.
SUMMARY OF THE INVENTION
[023] Accordingly, the present disclosure provides a dipstick for quick estimation of 15 glucose level in potato tubers stored at cold storage, the dipstick encompass a substrate impregnated with O-tolidinehydrochloride, Tartrazine, Triton X-100, and ethanol, and the impregnated substrate is further treated with Glucose oxidase, and Peroxidase.
[024] In another aspect of the disclosure provides a method of preparing the dipstick as outlined above. One more aspect of the disclosure provides a method for estimating 20 glucose level in potato tubers stored at cold storage,
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[025] The present disclosure is illustrated in figure 1 to 8 of the drawings accompanying this specification. In the drawings like reference number/letters indicate corresponding parts in the various figures 25
7
[026] Figure 1 represents colour developed by dipstick using known concentration of glucose ranging from 0 to 5000ppm.
[027] Figure 2 represents detection and comparison of glucose in potato tubers (after fresh harvest ˜ 110 days crop) (a): using dipsticks (b): using YSI (YSI Incorporated Life Sciences, Yellow Springs, Ohio) 2900 analyzer. 5
[028] Figure 3 represents detection and comparison of glucose in potato tubers stored at different temperatures[4°C, 12°C and ambient (15-25°C)]; a: using dipsticks, b: using YSI 2900 analyzer, c: potato chips colour
[029] Figure 4 represents detection of glucose using dipsticks stored for 15 days (one set at ambient temperature and another at 4°C). Standard glucose was used in the 10 experimentation.
[030] Figure 5 represents detection of glucose using dipsticks after one and half months of storage (one set at ambient temperature and another set at 4°C). Standard glucose was used in the experimentation.
[031] Figure 6 represents detection of glucose using dipsticks after five months of 15 storage (one set at ambient temperature and another set at 4°C). Standard glucose was used in the experimentation.
[032] Figure 7 represents Standard colour chart for glucose estimation as present in potato tubers (0 -5000 ppm).
[033] Figure 8 represents methodologies for preparation of potato sample for glucose 20 estimation. Method 1: by making a cut in the middle of the potato tuber. Method B: by slicing and extracting the juice from potato tubers. 1, 2, 3, 4, 5, 6, 7, 8 denote the step numbers.
25
8
DETAILED DESCRIPTION OF THE INVENTION
[034] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds 5 referred to or indicated in this specification, individually or collectively and any and all combinations of any or more of such steps or features.
Definitions
[035] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These 10 definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.
[036] The articles “a”, “an” and “the” are used to refer to one or to more than one 15 (i.e., to at least one) of the grammatical object of the article.
[037] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. Throughout this specification, unless the context requires otherwise the word “comprise”, and variations, such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated 20 element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.
[038] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
[039] The expression “%” is with respect to total weight % of the composition. 25
9
[040] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each 5 numerical value and sub-range is explicitly recited. For example, concentration of the component in the range of 0.01-5% should be interpreted to include not only the explicitly recited limits of 0.01-5%, but also to include sub-ranges, such as 0.01-1%, 1-3%, 3-5% 0.01-0.05% and , and so forth.
[041] Accordingly the present disclosure provides a dipstick for rapid estimation of 10 glucose level in potatoes tubers, especially potatoes stores at low temperatures.
[042] The method disclosed is a simple and quick method for glucose estimation in potato tubers, comprises of dipstick having components for estimation of glucose and a method for preparation of potato samples for glucose extraction and detection. Further the disclosure provides a standard colour chart developed for comparison of the 15 intensity of the colour development with the potato tuber samples.
[043] The present disclosure envisages the development of method for detection of glucose in tubers simply by cutting them and putting the strips in between two halves, pressed together or by extracting juice and dipping the strips to get a colour change. The developed strip contains dyes, enzymes and diagnostic base. The enzymes used 20 are capable of converting glucose to gluconic acid in the presence of oxygen and hydrogen peroxide, and another enzyme is capable of oxidizing dyes.
[044] In an embodiment of the present disclosure provides a dipstick for quick estimation of glucose level in potato tubers stored at cold storage, wherein the dipstick comprises of a substrate impregnated with O-tolidinehydrochloride in the range of 10 25 to 20mg/ml (weight by volume), Tartrazine 1 to 5 mg/ml (weight by volume), Triton X-100 0.001 to 0.010% (v/v), and Ethanol 0.001 to 0.010%. The impregnated substrate
10
is further treated with Glucose oxidase 25 to 35 mg/105 ml (weight/volume), and Peroxidase 10 to 20mg/ml (weight/volume).
[045] In another embodiment of the disclosure, the substrate for impregnating the above components is selected from a group consisting of filter papers selected from Whatman type filer paper, preferably CF3 type. 5
[046] Another aspect of the present disclosure related to a method of preparing dipstick for quick estimation of glucose level in potato tubers stored at cold storage, the method comprises of
a. preparing a impregnating solution comprising 1 to 3 parts by weight of Tartrazine, 12 to 20 parts by weight of O-tolidinehydrochloride, 0.001 to 0.010 parts 10 by weight of Triton X-100 and 0.001 to 0.010 ethanol, by dissolving O-tolidinehydrochloride and Tartrazine in distilled water and adding Triton X-100 mixed with ethanol, mixing the content thoroughly to get uniform solution,
b. impregnating a substrate by immersing the substrate in the above impregnating solution for 25 to 60 minutes and drying the substrate at a temperature in the range of 15 35 to 48oC for 15 to 90 minutes,
c. treating the above dried impregnated substrate with a solution of glucose oxidase and peroxidase dissolved in phosphate buffer solution at a pH in the range of 6 to 7.5 and drying the treated substrate at room temperature, and
d. cutting the treated substrate to required length and width and attaching one end 20 of the cut strip to a holder using non-reactive adhesive to obtain the dipstick.
[047] In another embodiment of the disclosure, the substrate for impregnating the above solution is selected from a group consisting of filter papers selected from Whatman type filer papers, preferably CF3 type.
[048] In an embodiment of the present disclosure, the cut-strips are stored in brown 25 color bottle with silica gel as desiccant at room temperature.
11
[049] One more embodiment of the present disclosure related to a method of estimation of glucose level in potato tubers stored at cold storage, said method comprises of:
a. contacting the dipstick as prepared about with a cut side of slice of potato or dipping the dipstick in a juice of cut and/or smashed potato for five to 30 seconds, 5
b. observing the color of the dipstick, and
c. comparing the developed color against a predetermined color chart having provided with glucose level against visual color, to estimate the glucose level of the potato tubers.
[050] In general, glucose levels below 0.1 percent (i.e. 1000ppm) on a fresh weight 10 basis are acceptable for chips and French fries production. The threshold levels of reducing sugars for making of French fries and chips is <150mg/100g Fresh weight and <100mg/100g Fresh weight, respectively.
[051] Since, the farmers growing potatoes for processing purposes does not know the reducing sugars levels before transporting the potatoes to, far off places/industries. In 15 addition, the farmers have no idea or clue whether their lot will be accepted or rejected because of high values particularly of glucose that affect the product quality of chips and French fries.
[052] The dipstick of the present disclosure helps the farmers in decision making before forwarding the tubers for longer distances. By using the unique dipstick and 20 knowing cut-off values of glucose in their lots (potato consignment), the farmers will be in a better position to know about whether their lot will be accepted or rejected in processing industry.
[053] In another embodiment of the present disclosure, safe limit of glucose level for potato processing has been estimated and the values are: 25
• 100ppm corresponds to 0.01%
• 250ppm corresponds to 0.025%
• 500ppm corresponds to 0.050%
12
EXAMPLES
[054] The following examples are given by way of illustration of the working of the disclosure in actual practice and should not be construed to limit the scope of the present disclosure in any way. 5
Example I
Preparation of impregnating solution:
[055] The impregnating solution is prepared as per the following formulation.
O-tolidinehydrochloride 12mg/ml 10
Tartrazine 1mg/5ml
0.05M potassium phosphate buffer pH 6.5
Glucose oxidase 3mg/ml. Prepare in potassium phosphate buffer pH 6.5
Peroxidase 1mg/ml. Prepare in potassium phosphate buffer pH 15 6.5
$Triton X-100 0.001% (v/v)
Ethanol 0.001% (v/v)
$- is a nonionic surfactant that has a hydrophilic polyethylene oxide chain (on average it has 9.5 ethylene oxide units) and an aromatic hydrocarbon lipophilic or hydrophobic 20 group. The hydrocarbon group is a 4-(1,1,3,3-tetramethylbutyl)-phenyl group.
[056] The following procedure was used for preparing the impregnating solution.
a. 72-mg of O-tolidine hydrochloride was dissolved in a 10-ml beaker and to this 6-ml double distilled water was added. This was dissolved thoroughly on vortex-mixer. 25
b. In another beaker, 5-mg of tartrazine in 25ml of double distilled water was dissolved. 30 mg of glucose oxidase was dissolved in 10-ml of phosphate buffer (pH 6.5).
13
c. Then in another beaker, 10mg of peroxidase was dissolved in 10-mlphosphate buffer (pH 6.5) and mixed properly.
Example 2
Preparation of reagent strips 5
[057] 1Four ml of above prepared O-tolidine hydrochloride solution, 2ml of tartrazine and10 μl each of triton X-100 and ethanol were taken in multi syringe boat. Filter paper strips (0.5cm width) cut into narrow strips (4-5 No.), were dipped for 45 minutes into the impregnating solution so that through the process of submersion and capillary action the strips are completely impregnated. It was kept for gentle shaking 10 on RockymaxTM shaker (Tarsons). The strips were then oven-dried at 42°Cfor 45 minutes. Dried strips were immersed in mixture of glucose oxidase and peroxidase (using mixture of equal volume) and dried at room temperature for 1-2 hrs. for complete drying. After complete drying, the developed strips of the reagent coated pad were pasted along the edge of photo glossy sheet (6 cm x 0.5 cm) using non-reactive 15 adhesive. The sheets were then cut into width of 5mm in such a manner that one end is free for handling. The cut strips were stored in amber colored bottles containing silica gel pouches as desiccant and stored at room temperature.
Example 3 20
Extraction of potato samples for glucose estimation
[058] The samples for glucose estimation can be prepared by various ways. The two methods/ways are as follows:
[059] Method 1: Cut the potato in two equal halves with knife or scalpel (figure 8(a)). Gently squeeze any one half of the cut potato and place the dipstick over the cut side 25 and keep it for 5-15 seconds until it is saturated by the potato juice. Let the dipstick dry for 1 to 2 minutes and then observe and compare the developed colour with standard colour chart.
14
[060] Method 2: Remove the peel of the potato and cut it into small slices and grind it with pestle and mortar or other suitable available means (e.g. grating). Transfer the juice to a small tube (leaving potato juice for settlement of starch; 1-2 minutes) and dip the dipstick in the juice for 2-3 seconds and take it out let it dry for 1 to 2 minutes and then observe and compare the developed colour with the standard colour chart (figure 5 8(b)).
Example 4
Detection of glucose in potato tubers using dipsticks
[061] In use, an impregnated strip is dipped into the liquid specimen (i.e. potato 10 juice/extract) to be tested by maintaining in contact with specimen from 5to 15 seconds. As a result, the distinct color intensities of progressively increasing amounts of oxidized dye is formed because of the presence of correspondingly increasing amounts of glucose in potato samples. The developed colours are clearly distinguishable by naked eye. The colour of the dipstick changes from original yellow 15 colour to light green to dark green and finally as black. When dipped in no glucose, the strips undergo no color change. Glucose was estimated in freshly harvested potato from Kufri farm of the institute (ICAR-Central Potato Research Institute) and used for estimation of glucose (Kufri Bahar, Kufri Jyoti, Kufri Pukhraj, Kufri Chandramukhi, Kufri Lauvkar, Kufri Chipsona-1, Kufri Chipsona-2, Kufri Chipsona-4 and 20 KufriFrysona) as well as in potato tubers stored at 4ºC, 12ºC and ambient temperature (Kufri Bahar and Kufri Chipsona-1). In case of storage experimentations, the potato varieties of Kufri Bahar and Kufri Chipsona-1 were harvested and stored at three different temperatures (4ºC, 12 ºC and ambient) for 120 days. After storage tubers were taken and used for glucose estimation. 25
[062] Refer to figure 2: Color developed from fresh harvest (after fresh harvest - 110 days crop) by obtaining glucose level (ppm) using standard test method YSI 2900
15
analyzer and dipstick of the present disclosure. From the figure 2, it is very clear that the results obtained are comparable.
Example 5
Preparation of color charts
[063] Standard color charts were prepared using standard glucose as well glucose 5 level obtained from potato tubers.
[064] Standard Color chart of standard glucose level developed by using dipstick of the present disclosure – figure 1.
[065] Standard Color chart of glucose level of potato tuber using standard method – figure 7. 10
[066] By comparing the colors at various ppm level of glucose i.e. 0, 100, 250, 500, 1000, 1500, 2000, 2500 & 5000 ppm, it was observed that both glucose from potato-tuber with convention method and glucose reagent with dipstick of the present disclosure provides identical colors and confirms the suitability of the dipstick of the present application. 15
Example 6
Analysis of storage life of dipsticks
[067] For testing the stability of developed dipsticks these were kept in small plastic vials and tightly closed. These packed dipsticks were kept at different temperatures 20 one at fixed 4ºC and other set at ambient temperature. At 15 day intervals these stored dipsticks were taken out and immediately used for glucose estimation. The stability of the strips were tested with standard solution of glucose having concentrations ranging from 100 ppm to 5000 ppm.
[068] Experiments were repeated as per the above method by storing the dipstick at 25 ambient temperature and one another set of dipstick stored at 4oC. Standard glucose was used for carrying the above experiment. The colors developed by using the
16
dipstick stored at two different conditions were compared. Referring to figure 4, it was found that colors were not exactly identical between the dipsticks stored at different conditions but the color trend (light yellow at 0 ppm, at 150 ppm darker color was observed, at 250 still darker color (yellowish green) compared to 150 ppm, at 500 ppm, ambient stored dipstick showed green color whereas the dipstick stored at 4 deg. 5 C showed lighter green. The same trend i.e. the dipstick stored at ambient temperature developed darker color compared to color obtained from dipstick stored 4deg C, up to 5000 ppm level. This confirms that the dipstick stored at two different conditions provides comparable color development.
[069] Similar results were obtained by using dipstick stored for one and half-months 10 at ambient temperature and at 4deg. Centigrade (figure 5); using dipsticks after five months of storage (one set at ambient temperature and another set at 4°C) (figure 6). In both experiments, standard glucose was used.
[070] The dipsticks were found to be stable and functional till five months of storage 4ºC and at ambient temperature. 15
[071] By carrying out number of experiments as outlined above, the applicant arrived at the following glucose level for safe limit and unsafe limit.
[072] Safe limit – up to 1000 ppm i.e. the potato tubers can be used for processing industry such as chips etc.:
• 100ppm corresponds to 0.01% 20
• 250ppm corresponds to 0.025%
• 500ppm corresponds to 0.050%
• 1000ppm corresponds to 0.1%
[073] Un- safe limit for potato processing: glucose level beyond 1500 ppm.
• 1500ppm corresponds to 0.15% 25
• 2000ppm corresponds to 0.20%
[074] The main advantages of the present disclosure are:
17
[075] A simple and quick method for estimation of glucose through dipsticks has been developed.
[076] Glucose estimation using the developed dipsticks and the kit thereof does not need any other accessories.
[077] Estimation of glucose can be performed in the lab as well on farm itself. 5
[078] Developed dipstick will avoid potato losses on transportation and chances of rejection at processor’s end.
[079] The developed dipsticks can be stored at a wide range of temperatures ranging from 4ºC to 25ºC for at least five months.
[080] Advantage of the present invention: Before transporting potatoes to industries 10 or to far-off places, travelling to long distances farmers can save their time, energy and money by checking their lots or material by using dipstick measuring glucose.
[081] The developed dipsticks are ready to use for glucose estimation in potato (specifically in the range of 0 to 2000 ppm required for differentiating the potato suitability for processing). 15
1. Dipsticks can be stored at low as well as ambient temperature.
2. Dipsticks can be used for detection of glucose in various other crops fruit, vegetables and cereals.

Claims:1. A dipstick for quick estimation of glucose level in potato tubers stored at cold storage, the dipstick comprises of a substrate impregnated with O-tolidinehydrochloride in the range of 10 to 20mg/ml (weight by volume), Tartrazine 1 to 5 mg/ml (weight by volume), Triton X-100 0.001 to 0.010% (v/v), and Ethanol 0.001 to 0.010%, and the substrate is further treated with glucose oxidase 25 to 35 mg/105 ml (weight/volume), and peroxidase 10 to 20mg/ml (weight/volume).
2. The dipstick as claimed in claim 1, wherein the substrate is selected from a group consisting of filter papers selected from Whatman type filer paper, preferably CF3 type.
3. A method of preparing dipstick for quick estimation of glucose level in potato tubers stored at cold storage, the method comprises of:
a. preparing a impregnating solution of 1 to 3 parts by weight of Tartrazine and 12 to 20 parts by weight of O-tolidinehydrochloride, 0.001 to 0.010 parts by weight of Triton X-100 and 0.001 to 0.010 ethanol, by dissolving O-tolidinehydrochloride and Tartrazine in distilled water and adding Triton X-100 mixed with ethanol, mixing the above to get uniform solution,
b. impregnating a substrate by immersing the substrate in the above impregnating solution for 25 to 60 minutes and drying the substrate at a temperature in the range of 35 to 48oC for 15 to 90 minutes,
c. treating the above dried impregnated substrate with a solution of glucose oxidase and peroxidase dissolved in phosphate buffer solution at a pH in the range of 6 to 7.5 and drying the treated substrate at room temperature, and
d. cutting the treated substrate to required length and width and attaching one end of the cut strip to a holder using non-reactive adhesive to obtain the dipstick.
4. The dipstick as claimed in claim 1, wherein the substrate is selected from a group consisting of filter papers selected from Whatman type filer paper, preferably CF3 type.
5. A method as claimed in claim 3, wherein the cut-strips are stored in brown color bottle with silica gel as desiccant at room temperature.
6. A method of estimation of glucose level in potato tubers stored at cold storage, said method comprises:
a. contacting the dipstick as claimed in claim 1 with a cut side of slice of potato or dipping the dipstick in a juice of cut and/or smashed potato for five to 30 seconds,
b. observing the color of the dipstick, and
c. comparing the developed color against a predetermined color chart having provided with glucose level against visual color, to estimate the glucose level of the potato tubers.