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Dabigatran Monitoring Was Influenced by Thrombin Time Reagent With Different Thrombin Concentrations

To describe the effect of dabigatran on thrombin time (TT) reagents at different concentrations of thrombin. Pooled normal plasma enriched with dabigatran was dissolved in dimethylsulfoxide (DMSO) at concentrations of 0, 20, 50, 100, 200, 300, and 500 ng/mL. Samples with each concentration were evaluated using a semiautomatic coagulation analyzer to assess the effect of dabigatran on internal normalized ratio (INR), thromboplastin time (APTT), and TT, which were purchased from Instrument Laboratory (IL), Sysmex (SYS), and Stago (STA), respectively. Regarding INR, no reagent showed good sensitivity to increasing concentration of dabigatran, despite all reagents showing good linear response curves (P = .012). Regarding APTT, all reagents had low sensitivity to increasing dabigatran concentration, but SYS-APTT showed a better linear response curve (P = .001). Regarding TT, all reagents had a good linear response to the concentration of dabigatran; however, SYS-TT was very sensitive at low concentrations of dabigatran (0-100 ng/mL), while IL (TT-5 mL) and STA-TT were sensitive at medium concentrations of dabigatran (0-300 ng/mL), and IL (TT-2 mL) was less sensitive for a wide concentration of dabigatran (0-500 ng/mL; P = .007). Internal normalized ratio and APTT showed low sensitivity and SYS-TT showed high sensitivity to concentrations of dabigatran that were unsuitable to monitor. Both IL (TT-5 mL) and STA-TT were useful at medium concentrations of dabigatran by semiautomatic coagulation analyzer, which calculated results using the end point method of coagulation. Instrument Laboratory (TT-2 mL), which contains a higher concentration of thrombin, had better sensitivity to the concentration of dabigatran than APTT and was suitable for routine monitoring by an automatic analyzer.

Introduction
Dabigatran etexilate is an oral direct anticoagulant and a direct thrombin inhibitor. 1,2 Dabigatran is frequently used to prevent nonvalvular atrial fibrillation, thromboprophylaxis in knee and hip replacement surgery, and for the treatment as well as secondary prevention of venous thromboembolic disease. 2 Dabigatran etexilate has a wide therapeutic window and can be pharmacokinetically assessed without any kind of monitoring. 3 However, there are several clinical situations where determination of the level of anticoagulation may be of value, such as patients with massive hemorrhage or thrombosis, or excessive anticoagulation treatment. 2 In addition, reasonable monitoring (thromboplastin time [APTT]/thrombin time [TT]/ecarin clotting time [ECT]) can ensure blood concentrations of dabigatran within the normal range before operation, especially of patient with renal impairment. 4 There are many coagulation screening assays to monitor dabigatran, such as APTT, prothrombin time (PT), TT, diluted thrombin time (dTT), and ECT. Ecarin clotting time and dTT are sensitive to the blood concentration of dabigatran; however, they are not routine laboratory screening assays. 2,5 Dager et al 6 found that APTT and internal normalized ratio (INR) showed a linear response to dabigatran concentration without enough sensitivity, and that TT was extremely sensitive to the changes in dabigatran concentration, but they did not have enough data to analyze TT because of the limitations of the detection range in automatic coagulant analyzers. The basic reaction principle for PT, APTT, and TT is blood coagulation. However, analyzers have different methods to generate data, for example, analyzers from SYS and Instrument Laboratory (IL) obtain the results via immunoturbidimetry, whereas the analyzer from STA uses coagulation as the end point (Golden Standard). Due to these method differences and the variation of detection levels among analyzers, the correlations between dabigatran's plasma levels and their impact on clotting assays remain unclear. The semiautomatic coagulation analyzer (STA-R Evolution) is quite different. In contrast to automatic coagulation analyzers, STA-R, which obtains results using the end point method of coagulation, can detect the result over a long range of time, allowing the selection of better reagents for monitoring dabigatran.
The half-life of blood dabigatran in adults is reportedly 14 to 17 hours; in healthy males receiving a single 150 mg dose, the peak blood concentration is 90 to 130 ng/mL, which is maintained for 1 to 4 hours. 7 The RELY Trial indicated that the blood concentration of dabigatran in 80% of patients was 22.9 to 238.3 ng/mL. We prepared several samples with concentrations of dabigatran between 0 and 500 ng/mL, and we aimed to analyze the results of APTT, TT, and INR using a semiautomatic coagulate analyzer. We then aimed to further elucidate the relationship between the thrombin concentration of different TT reagents and blood concentrations of dabigatran.

Materials and Methods Materials
Dabigatran etexilate (Boehringer Ingelheim International GmbH, Ingelheim am Rhein, Germany) was bought from a pharmacy. Pooled normal plasma was provided by Stago (Lot No: 00539, Diagnostica Stago, Asnière sur Seine, France). Dimethylsulfoxide was obtained from Sagan Corporation (Shanghai, China). The core reagents (PT, APTT, TT) were obtained from the 3 companies: SYS from Japan, STA from France and IL from United States. Importantly, the TT reagent from IL has 2 concentration of thrombin (IL-5 mL: 3.0 UNIH/ mL and IL-2 mL: 7.5 UNIH/mL). The thrombin concentration of SYS-TT was 1.15 UNIH/mL and the thrombin concentration of STA was 1.5 UNIH/mL. The STA-R was provided by STA.

Methods
Samples with different concentrations of Dabigatran. Pooled normal plasma was dissolved in 1-mL distilled water at room temperature for 30 minutes. A capsule of dabigatran etexilate with the capsule shell removed was dissolved in 2.2 mL of dimethylsulfoxide to form the stock solution (50 mg/mL). Prepared mixed plasma was diluted to final concentrations of 0, 20, 50, 100, 200, 300, and 500 ng/mL. Statistical analyses. The relationships between the results of reagents from different companies and dabigatran concentrations were expressed by the Pearson correlation analysis. The coagulation assays were compared using the Friedman test, with P < .05 considered to be significant. Data analysis was performed using SPSS.

Results
The reagents PT, APTT and TT, selected from 3 different companies and the STA-R Evolution, were employed to assay the relationship between coagulation test items and the blood concentration of dabigatran. We evaluated the related data from Table 1.

Internal Normalized Ratio
The results of INR show that the STA-PT was more sensitive to the changes in the blood concentration of dabigatran than SYS-PT and IL-PT ( Figure 1). All reagents had a good linear response curve. The distribution of INR results from different companies with increasing concentration of dabigatran was significantly different (P ¼ .012).
Thromboplastin Time Figure 2 shows that the STA-APTT reagent had the best sensitivity and correlation with changes in the blood concentration of dabigatran. The IL reagent sensitivity was medium and SYS reagent sensitivity was lower, but its correlation was better than that of IL reagent. The distribution of APTT results from different companies with increasing concentration of dabigatran was significantly different (P ¼ .001).

Thrombin Time
Instrument Laboratory-TT reagents include TT-5 mL and TT-2 mL, representing different thrombin concentrations. Table 1 shows that TT-5 mL and STA-TT could detect concentrations of dabigatran within 300 ng/mL, whereas TT-2 mL could detect concentrations of dabigatran within 500 ng/mL, and SYS-TT could detect concentrations of dabigatran within 100 ng/mL. Figure 3 shows that SYS-TT is most sensitive to changes in the blood concentration of dabigatran, with TT-5 mL and STA-TT being medium sensitive and then TT-2 mL being least sensitive. Notably, the 4 reagents showed good linear correlation. The distribution of TT results from different companies with increasing concentration of dabigatran was significantly different (P ¼ .007). In a side-by-side comparison of the 3 IL detection methods (Figure 4), APTT sensitivity was low and TT-5 mL was very sensitive; when the blood concentration exceeded 300 ng/mL, the detection value was greater than 400 seconds. Additionally, IL (TT-2 mL) could detect the blood concentration within 500 ng/mL with medium sensitivity. All parameters showed good linear correlation.

Discussion
The standard oral dosage of dabigatran etexilate is 110 mg or 150 mg, twice per day. The blood concentration of dabigatran is stable without routine coagulation monitoring. 8     function and increased risk of bleeding, some hospitals of European countries have chosen to use 75-mg dabigatran twice daily. 9 Therefore, monitoring of the blood concentration of dabigatran is critical for some patients. Although ECT and dTT are suitable for the monitoring of dabigatran, they are not recognized by the United States Food and Drug Administration; thus, they are not widely used for clinical monitoring. 5 William et al 6 stated that although INR and APTT showed a good linear relationship with the blood concentration of dabigatran, their sensitivity was poor, and it was unclear whether these parameters could be used for monitoring. William et al randomly sent prepared blood samples to laboratories in South America and the United Kingdom for testing and statistical analysis. Considering the inconsistent results by different analyzers, some interference caused by delivering samples, and the narrow range of blood concentration of dabigatran, we have made some improvements to make clear relationship between reagents and dabigatran. First, we chose reagents from 3 companies popularly used in clinical thrombosis and hemostasis laboratories. Second, we applied the semiautomatic coagulator STA-R as the detection analyzer. The STA-R can detect the result over a long range of time at higher concentrations of dabigatran and using the end point method of coagulation potentially eliminating some detection interference. Finally, we prepared samples with a wide range of blood concentrations of dabigatran and analyzed them within the suitable time and temperature.
Internal normalized ratio was less sensitive to blood concentrations. Stago-INR increased by 0.1 to 0.3 per 50 ng/mL and was less sensitive to low blood concentrations. If the blood concentration of dabigatran had no large shift, it was very hard to recognize changes of the results. Van  measurements were not helpful for dabigatran monitoring, whereas APTT was appropriate. In a comparison among the 3 APTT reagents, STA-APTT had the highest sensitivity and showed a good linear relationship with the blood concentration of dabigatran (P ¼ .001). Dubé et al 11 compared the sensitivity and specificity of 5 different APTT reagents with TT and dTT reagents. Although APTT showed good sensitivity, its specificity was very poor for low blood concentrations. So APTT is not a good choice in monitoring the blood concentration of dabigatran.
The semiautomatic coagulation analyzer was better in assays, resulting in high TT results. Sysmex-TT was highly sensitive: when the blood concentration of dabigatran exceeded 100 ng/mL, the detection value was >400 seconds. The SYS-TT reagent, which contained thrombin at approximately 1.15 UNIH/mL, suffered the greatest interference by dabigatran, which was at the lowest concentration among 4 TT reagents. The sensitivity and correlation measurements of IL (TT-5 mL) and STA-TT were similar. When the blood concentration reached 500 ng/ mL, the detection values were >400 seconds. However, the thrombin concentrations of IL (TT-5 mL) and STA-TT were 3.0 UNIH/mL and 1.5 UNIH/mL, respectively. As shown in Table 1, the results of IL (TT-5 mL) were lower than those of STA-TT in low blood concentrations of dabigatran, which were determined by residual thrombin concentration. When the thrombin concentration increased, the difference of TT results between them was not obvious, and the detection values exceeded 400 seconds when the concentration reached 500 ng/mL. In comparison, IL (TT-5 mL) with a high thrombin concentration was optimal for monitoring. With the increase of blood drug concentration of dabigatran, IL (TT-2 mL) retained a high correlation, and the sensitivity was much higher than APTT.
According to the automatic coagulation analyzer, Avecilla et al 12 prepared dTT reagent, which contains 0.75 UINH/mL of thrombin, to reduce sensitivity to dabigatran. Compared to APTT and TT, dTT showed monitoring advantages. The RELY Trial indicated that the blood concentration of 10% to 90% dabigatran was 22.9 to 238.3 ng/mL. The upper limit of TT detection of SYS and STA analyzers is approximately 200 seconds, whereas that of the IL analyzer is 300 seconds. Instrument Laboratory-2 mL contained a thrombin concentration of 7.5 UINH/mL, the residual thrombin would have a reaction with prothrombin after combining with dabigatran, but the TT results had a good sensitivity and correlation with the concentration of dabigatran. Using various coagulation reagents for detection on a semiautomatic coagulation analyzer and using the end point method of coagulation to interpret the results, this study facilitated analysis of the differences of various reagents. The end point method of coagulation remains the golden standard. The semiautomatic coagulation analyzer is suitable for anticoagulant monitoring when an automatic coagulation analyzer cannot meet the requirements for anticoagulant monitoring.
Importantly, it is difficult to obtain samples of patients with different blood concentrations of dabigatran; thus, this was an  in vitro test, and it is not clear whether dabigatran monitoring and potential dose adjustments have any impact on clinical outcome. However, it is meaningful to monitor the blood concentration of dabigatran in consideration of thrombin concentration from different TT reagents. As some actual situations in the laboratory, and we did not consider the problem of lot-to-lot variability, we obtained reagent with one lot number to measure each blood concentration of dabigatran, however, we tested each blood concentration of dabigatran for 3 times, the results of our study could reflect potential outliers.

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

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Formulation of gluten-free cupcake with hydrocolloids for people with Celiac disease

Background: The development of gluten-free products is a great challenge for manufactures aiming to make baked products have a similar composition to their gluten-containing counterparts. They routinely use commercial and vegetable additives (rice flour and cassava starch), which are gluten free. Due to the fact that most of these products in Venezuela are imported and thus expensive, the prominence of celiac disease increases by 4% every year. This work aimed to develop a filled cupcake formulation with the use of natural and synthetic gluten-free hydrocolloids for those with celiac disease. Methods: Two mixture designs, one with rice flour and one with cassava flour, were examined to identify which flour would give the cupcake the best consistency. The flours were mixed with hydroxypropyl methylcellulose (HPMC) and vegetable fat (emulsifier) to see which proportion of ingredients was best-suited for the final product. The response variables of adhesiveness, cohesiveness, hardness, and gumminess were used to develop a texture profile. Two formulations were then obtained based on desirability ratings, and then evaluated based on the acceptability of 50 celiac patients. The physical, chemical and microbiological characterization, along with the storage stability of the selected formulation, was evaluated. The shelf life of the cupcake was determined by evaluating the texture profile, acceptability and microbiological parameters during 21 days at three temperatures (25 °C, 35 °C and 45 °C). Results: The formulation with the greatest desirability in our population had the lowest gliadin content (0.231ppm), the lowest production cost, and showed a decrease in acceptability as time and temperature increased. The shelf life of the cupcakes was estimated to be 14 days when stored at an average temperature of 28 °C. Bioactive Compounds in Health and Disease 2019; 2(6): 134-148 Page 135 of 148 Conclusion: Our study demonstrates that it possible to produce a low-cost, high-calorie filled cupcake for people with celiac disease to consume.

The replacement of gluten in baked products is one of the great challenges for those working in food science. Patients with celiac disease, or gluten-sensitive enteropathy (GSE), must adhere to a gluten-free diet for life. To make quality gluten-free products, hydrocolloids are used due to their ability to change the rheological properties of the product, thus improving its texture [5].
A range of gluten-free bread (GFB) formulations have been developed using rice and maize flours. These flours are often combined with maize, potato, or cassava starches because they are widely available, inexpensive and bland in taste and flavor.
In Venezuela, these specialized products are generally imported and of high cost because their availability depends on several external factors: import procedures, currency settlement, availability of resources, nationalization of the product, marketing permits, among others. In addition, there is a limited variety of these products because the market is small, manufacturers are scarce and there is little competition [6]. Therefore, this research was conducted to develop a filled cupcake formulation with the use of natural and synthetic gluten-free hydrocolloids for those with celiac disease.

Selection of ingredients
A homemade recipe of a normal cupcake was selected to determine the ingredients. The glutenfree hydrocolloid ingredients, HPMC, CMC, Monopals and, Xanthan, were substituted for the wheat flour. These ingredients were intended to improve the texture of the product. The recipe selected to make the cupcake is shown in table 2.

Preliminary tests
Due to the variety of hydrocolloids in the market, preliminary tests were done to examine which ones worked best. The limits of each hydrocolloid were set by their respective suppliers (Table 3). Once the hydrocolloids were obtained, two D-optimal mix designs were made. The first design had a 27% variation of the formulation, and contained the following ingredients: cassava flour, HPMC and Monopals (Table 4). The second design, with a variation of 30%, had the following ingredients: rice flour, HPMC and Monopals (Table 5). Subsequently, 18 formulations were obtained for each of the designs. The response variables, hardness, adhesiveness, cohesiveness and gumminess, were determined with the use of a texturometer (TA.XTplus, Stable Micro Systems). A texture profile analysis (TPA) was established by the standard method N° 74-09 [7]. This test reports the force used to compress the product by 25% of its height and the force required to maintain compression for 60 s [8].

Optimization of the formula
Finally, an optimization of the formulation was done using the function of desirability. This was found using the values of the response variables for a wheat-flour cupcake (control). Using this function, the two formulations were sensory characterized.

Quantitative verification of the presence of gluten
The presence of gluten was determined using the immunoenzymatic sandwich assay (ELISA) to quantitively analyze the prolamins of wheat (gliadin), rye (secalin) and barley (hordein) in foods declared free of gluten. This was done using the Diagnostic Automaiton, Inc. (DAI) Gliadin / Gluten ELISA kit. This method is based on the detection of proteins by the antigen-antibody reaction. The amount of color is proportional to the number of projects in the sample, which was measured by a microplate spectrophotometer (TECAN).

Sensory characterization for the selection of the formula
The acceptability was tested by a panel of 50 people, all of whom had celiac disease or a related condition. In addition, the statistical analysis Check All That Apply (CATA) was performed in order to obtain descriptors of the gluten-free cupcakes.

Determination of stability during storage
The stability during storage of a food product helps to determine the product's shelf life, the time a product can be stored before being consumed. The microbiological parameters of molds and yeasts were measured in the cupcakes and compared to the Venezuelan standard [9]. In addition, the TPA, as previously described, was calculated. A value of acceptability was also obtained through the use of a structured hedonic scale of nine points (9 = I like very much, 5 = I am indifferent and 1 = I dislike a lot). The stability during storage was performed for a period of 21 days, and the product was stored at three different temperatures (25 ± 2, 35 ± 2 and 45 ± 2 °C).

Statistical analysis
The validity of the variance analysis was performed for each response variable of the D-optimal mixing design. Three assumptions were verified for each response variable: normality, constant variance and residency independence. Once each assumption was verified, the significance of the model was in turn verified and not adjusted any further.

Selection of ingredients
After choosing the recipe, preliminary tests were performed to select the optimal gluten-free ingredients. The baking height and specific volume (SV) of the formulations were evaluated. A high specific volume is desired, because with such there are more interactions between the molecules of the product, thus improving its texture quality [10].
As shown in Table 7, the products made with HPMC and monopals were taller and had greater specific volumes in comparison to the products made with CMC and Xanthan. As a result, the hydrocolloids HPMC and monopals were selected to makeup the mix design in addition to the natural hydrocolloids of rice and cassava flour.  Table 11 exhibits the texture profile values of each response variable for the rice flour formulations. Table 8. Response variables of D-optimal mixture design with 30% variation of the formulation.

Formula optimization
The desirability function suggested three formulas, from which two within the optimum design area were selected. The contour of the optimization zones is show in Figure 1.   The hardness values of the cassava flour formulations were significantly higher than those of rice flour. This increased hardness results in an undesired texture of the cupcakes.

Proportion of components Response variable
The higher values of hardness in cassava flour can be attributed to the retrogradation of the starch. The polysaccharide is composed of amylose and amylopectin, in which the latter's structure changes when it is saturated by water molecules. The amylopectin transitions from an amorphous state to a vitreous one, causing the progressive increase of hardness in the cupcakes [11]. Excessive hardness and gumminess can also be caused by the elastic properties of the hydrocolloids. The texture of the product can vary when mixed with starches from different sources. Based on the given information, the rice flour formulations were selected.

Determination of acceptability
As previously stated, the acceptability of a regular (gluten-containing) cupcake was compared to that of the two gluten-free formulations using a hedonic scale of 9 points. The acceptability was rated by 50 people with celiac disease or a related condition.
The acceptability values can be seen in Table 11. It is notable that while formulation 1 had a higher acceptability value than formulation 2, the difference was not statistically significant (p>0.05). Since the acceptability values were similar, the selection of a final formulation will depend on other factors. It is also notable that both of the gluten-free formulations had a lower acceptability value than that of the cupcake with gluten.

Descriptors obtained through the analysis of CATA
A Factorial Correspondence Analysis (FCA) was performed to examine the relationships among the descriptors found through CATA (Figure 2). The gluten-free formulations are located to the left of the F1 coordinate, while the control formulation is located to the right of it. In comparing the two gluten-free formulations, formulation 2 was associated with more flavor descriptors than formulation 1. Regarding texture, though, formulation 1 was more closely associated to being spongy, which is an admirable characteristic in baked products.

Determination of gluten
The only treatment for those with celiac disease is to strictly adhere to a gluten-free diet, abstaining from wheat, oats, rye and barley. Hence why it is so important to find gluten-free foods that not only taste good but have nutritional value as well. Those with celiac disease cannot consume any food that has been contaminated with gluten at any point in time from its origin to its final commercialization [13]. The gliadin and gluten concentrations of both cupcake formulations can be seen in Table 11.
According to the results obtained by the ELISA sandwich enzyme-immunoassay method, both formulations contain less than 20 ppm gluten. As a result, they can be labeled as being "glutenfree," as established in the Codex Alimentarius. As long as there is no cross-contamination in the preparation of the cupcakes, they are suitable for the consumption of people with celiac disease.

Selection of the formulation
After reviewing the above information, formulation 1 was selected for a variety of reasons. These included having a desirability rating close to 1 (0.81), being described as spongy, and having a lower production cost than formulation 2.

Product stability Microbiological parameters during stability storage
The quantification of molds and yeasts in food is very important, as it can be used to indicate adequate or inadequate sanitary practices during the production and storage of products. It can also be used to examine the quality of raw materials. The microbiological parameters, mold and yeast, of the filled cupcakes can be seen in Table  13. For the first three weeks of the study, there was less than 10 cfu/g of molds at 25 °C and 45 °C. In the first two weeks of the study, there was less than 10 cfu/g of yeasts at all three temperatures (25 °C, 35 °C, 45 °C). These results are within the parameters established by the Venezuelan standard. It is notable that until the third week, the mold and yeast counts were low, inferring that good hygienic and sanitary methods were used in the preparation of the mixture. After 21 days of storage at 25 °C though, an increase in yeast was observed. This can be attributed to the fact that this temperature, in addition to a decrease in pH, generated optimal conditions for yeast growth [14].

Texture Profile Analysis (TPA) during storage Hardness during storage.
The hardness of a product, measured by a texturometer, is defined as the peak force that is achieved during the first compression cycle. In other words, it is the resistance that prevents a permanent deformation. The phenomenon of hardness in cupcakes has scarcely been studied, but it is known that the complex interaction between multiple ingredients affects the texture of the final product [15]. The hardness of the filled cupcakes can be observed in Figure 3. As time went on, an increase in hardness was observed. However, this increase was more evident at 35 °C and 45 °C. This can be attributed to the process of food deterioration known as staling. In this process, the moisture of the cupcake is re-distributed, giving it a hard and smooth consistency. This process is favored at high temperatures, which explains the results of the cupcakes examined [16].

Cohesiveness during storage
Cohesiveness is the mechanical property of texture that indicates how well the form of particles is maintained after force is applied. It is due to the nature and concentration of the material in the intercellular spaces of a product [17]. The cohesiveness of a food represents the magnitude of deformation after being broken by the application of force.
As seen in Figure 4, the cohesiveness of the products in the first week was similar at all three temperatures (25 °C, 35 °C, 45 °C) (p>0.05). At all three temperatures, the cohesiveness increased up until 15 days of storage. At this point, the cohesion of the cupcakes at 25 °C slightly decreased, while the cohesion at 35 °C and 45 °C increased at a higher rate.

Gumminess during storage
The gumminess of a product is calculated by multiplying the hardness and cohesiveness values of the given product. Therefore, increasing these two factors would increase the gumminess of a product.
As seen in Figure 5, the gumminess of the cupcakes was relatively low until days 8-9 of storage. At roughly 14 days, the gumminess of the cupcakes at 25 °C remained relatively constant, while that of the cupcakes at 35 °C and 45 °C increased greatly (p<0.05).

Cohesiveness
Firmness Time (t) Figure 5. Variation of the gumminess as a function of time and temperature.

Evaluation of adhesiveness during storage
Adhesiveness is the degree to which a food sticks to a given surface. Products that contain hydrocolloids are generally more stable than gluten products when kneading. This is likely due to the high degree of internal cross-linking seen in hydrocolloids due to the formation of disulfide bridges. As a result of this viscoelastic network forming, it is probable that the adhesiveness of the cupcakes will be greater during the first weeks of storage [12].
As seen in Figure 6, the adhesiveness of the cupcakes progressively decreases over time at temperatures of 35 °C and 45 °C, while it increases slightly and then decreases at 25 °C. It is also notable that at day 14 of storage, the adhesiveness of the samples at all three temperatures was the same (p>0.05).

Acceptability during storage
The acceptability of the cupcakes was examined during storage (Figure 7). For the first two weeks, the minimum value of 5.3 was seen at 45 °C after 14 days, while the maximum of 7.5 was observed at 25 °C when the storage test began. These two values correlate to "I am indifferent" and "I like moderately," respectively. However, after 21 days of storage, the minimum acceptability value dropped to 4.2 at a temperature of 45 °C , while the maximum value was 6.0 at 25 °C. These two values correlate to "I dislike slightly and "I like slightly," respectively.
The level of intent to purchase the cupcakes was examined as well. During the first few days of storage, the maximum intent to purchase of 90% was seen in the cupcakes stored at 25 °C, while the minimum of 85% was seen in the cupcakes at 45°C. However, as the time of storage increased, the intention to purchase decreased. After 21 days of storage, the maximum intent to purchase was 63% for the cupcakes stored at 25 °C, and the minimum was 49% for the cupcakes stored at 45 °C.
The decrease of acceptability in the cupcakes can be attributed to the chemical deterioration of the baked products caused by hydrolytic and oxidative rancidity processes. These processes cause unpleasant odors and flavors, hence why the panelists disliked the products [18].

Estimation of shelf life
To determine the shelf life at 28 °C, the average temperature in Venezuela [19], Log t vs. T (°C) was plotted to obtain an equation of time as a function of temperature ( Figure 8). It was found that at 28 °C, the product can last 14 days without undergoing significant changes in hardness (R 2 = 0.83).
It is observed that there is an inverse relationship between temperature and the shelf life of the product. In the literature examined there was no standard shelf life of baked goods due to the fact that the hardness of a crumb varies according to the constituents, additives and temperature [20].

CONCLUSIONS
A low-cost gluten-free formulation of a filled cupcake, with high acceptability, was selected. There was an inverse relationship between both time and temperature and the acceptability of the cupcakes as rated by possible consumers. In addition, the texture of the cupcakes deteriorated as time went on. Due to the processes of aging and rancidity, the product became tough, gummy, and less adhesive. Finally, the shelf life of the filled cupcake, when kept at the average temperature of Venezuela (28 °C), was 14 days.


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Production data of wine grape gene bank (Vitis spp.) of University of Debrecen, east Hungary

National wine strategy of Hungary promotes the use of “flexible” grape cultivars. These enable producers’ best fit to wine market changes and expectations. This study is aimed to present data on the gene bank of the University of Debrecen, Hungary. Data were collected at a single site, between 2010 and 2018 in east Hungary lowland on acidic sandy soil, own rooted planting material. Our results showed that besides high yield and adequate cane production desired sugar content at convenient pH is to be awaited with moderate deviation between vintages. Presented concept demonstrate technological flexibility of cultivars by their average deviation from regression equation between increasing sugar and pH typical for the vintage composed of data of cultivars of the gene bank. Average positive deviation means higher sugar content at specific pH, thus higher sugar content at desired, conveniently low pH (3.0-3.2 pH).

Introduction
National wine strategy points out quality wine production. This is the strength of Hungarian wine production sector. Mass production is not an option in international market concern. Another aim of the strategy to focus on "flexible" cultivars. This means that a circle of cultivars could be defined technologically flexible rendering possibilities for growers and wine makers to better fit to wine-market (for example: 'Kékfrankos', 'Rizling', 'Furmint'). Production goal can greatly vary, but there is a general need for a portfolio of products basing financial safety of the enterprise. Possibilities could be backed by data on biological productivity potential of the cultivars. This paper is to disseminate data of the variety collection of University of Debrecen from this perspective.
Earlier papers focusing on resistant, interspecific (PIWI) cultivars (Rakonczás, 2011(Rakonczás, , 2015 detail technological possibilities and strength of this innovative group. The importance of these varieties (and hybrids) is considerably increasing due to very expansive effects of globalization and climate change. These highly affect vine growing-and wine making technologies. Producers are increasingly forced to focus on the market. Biological potential and resistance to pests and climatic extremities, thus also ecological adaptability of cultivars became crucial information.
Biological productivity of cultivars could be primitively demonstrated with average yearly harvest data. Ravaz-index encomprise data of yearly cane production (Ravaz, 1903). There is a general rule, that higher quantity equals lower quality. It is important to comprehend, that this rule in this wording is very rough. System of sink-source relations is more sophisticated and highly more sensitive with its physiological background (Csepregi, 1982, Ribereau et al., 2006, Keller, 2000, Lőrincz & Barócsi, 2010, and market expectations (problematics of rosé wine). Ravaz-index (Ravaz, 1903) can greatly deviate, according to the vegetative or generative character of the variety, to applied phytotechnical practices and the condition of the plantation. It is important to respect that the derived data hides the real total biological productivity (10kg/5kg=2 and 2kg/1kg=2).
In this context Tomcsányi & Német (1963) cit. Csepregi (1982 state that a variety is to be deemed valuable, of which the cane production between vintages does not show great variation, and in comparison to other cultivars a considerably higher yield is harvested on the same level of cane production. A criteria is to be added: besides higher and stable yield with adequate and stable cane production (Német, 1963, Csepregi, 1982 the desired sugar content is reached besides adequately low pH (Rakonczás, 2015).

Materials and methods
The variety collection of the University of Debrecen was established in Pallag, on immune sandy soil, by 3 m between row and 1m between stock spacing trained for single curtain stock form, with the use of European own rooted planting material. Five stocks of each cultivar represent one experimental block.
Nutrition is carried out on the basis of the specific nutrient demand of the grape (Kozma, 1993) giving out 310 kg NPK (effective material) manure on yearly bases in two phases. More detailed description was given in an earlier paper (Rakonczas, 2015). Because of considerable presence of ESCA, this work represents our last published data collection in this topic.
The following data were collected: yield (kg/stock), cane production (kg/stock), of which the (Ravaz-index, or Y/N-ratio (Ravaz, 1903)) the use-up index of wood yield is calculated (kg yield/kg cane). Sugar content and acidity at harvest is presented according to the introduced concept.
Data were processed with Microsoft Excel. Besides average data of 2010-2018, CV is also given: CV= ((Deviation / Average of vintages) * 100). This is to demonstrate technological-, climatic robustness of the cultivars.

Results and discussion
It is clear to comprehend that besides adequate yield and cane production, better technological flexibility of the variety could be detected in phenomena, when increasing sugar content meats lower level of acidic breakdown (less steep pH increase) ( Table 1). By increasing sugar content at ripening, pH consequently increases, since titratable acidity lowers (Marcus, 2000;Ribéreau et al., 2006). On average, producer could estimate an increase of about 1.5-2.0 sugar degree a weak. In optimal case 18-20 Hungarian sugar degree (180-200 g/litre) is reached at 3.0-3.2 pH. Within variety categories in each vintage regression equation was put on data of varieties of the collection. Deviation of single variety data from regression line shows, whether sugar accumulation of specific cultivars at a given pH is lower or higher than the average. The best case is to get higher sugar degree at lower pH, in case of corresponding high yield and adequate cane production. This demonstration does not content data of 2014 for abnormal rainfall and berry rot, and 2016 for problems of pH measurement. In case of red wines varieties 2016 is also missing, because few data could be collected. From the point of mass production the following white wine grape cultivars are to be highlighted (Tables 2-3): 'Cirfandli', 'Generosa', 'Aletta', 'Göcseji zamatos' (high yield and cane production). It is to be mentioned, that nearly all interspecific varieties show higher values both in yield and cane production. From the point of discussed concept, these data demonstrate the vigour and production potential of specific cultivars.
On second step, deviation of sugar accumulation compared to regression equation line at a given pH (decision of wine making technology) demonstrate adaptability of the specific variety to the concept of technological flexibility. Levels of CV mark between-vintage stability of the data (the lower the better). Presented data suggest, that qualified 'Ezerfürtű', 'Generosa', 'Korona', 'Pátria', 'Sauvignon', 'Zengő', 'Zéta', 'Zeus'; from the circle of candidates 'Gyöngyrizling', 'Heureka' and 'Tarcal 1 and 7' represent perspective choice. From the circle of interspecific (PIWI, or innovative, resistant group) cultivars 'Kunleány', 'Odysseus' and 'Viktoria gyöngye' show perspective data.
Set of production data of red wine grape cultivars does not comprise any extending case ( Table 4). Cane production does not show in-balanced vegetative-generative harmony either. It is due to note, that other type of pruning and training strategyinstead of single curtain -would probably facilitate higher production levels. Only 'Cabernet sauvignon', 'Alicante Bouchet', 'CsV525', 'Dornfelder' and interspecific 'Duna gyöngye' (Perla de Danube) could probably be highlighted.
Concept of flexibility can not be shown in case of red wine cultivars, because data bias (ESCA, etc.) and inconveniences of the training system. Data of cultivars of good reputation like 'Kékfrankos' (Blaufrankish) gives an example for this constraint.

Conclusions
The presented concept could give a guideline in evaluation of wine grape cultivars. In integrated production each element of the technology should aim at production policy, and support marketing. Plant protection and plant condition (capacity and health of tissues) is also essential in quality wine production besides discussed production data and basic quality parameters. In an ideal plant condition pattern and structure of the plantation, besides appropriate training system and pruning strategy, modification of technological elementsmost of all load of the stacks, fitotechnical measures (Lőrincz & Barócsi, 2010)considerable flexibility can be achieved. Highlighted cultivars of this paper give suggestion to chose the best appropriate cultivars of better flexibility.  Table 3. Production and flexibility data of candidate Vitis vinifera (L.) and intespecific (qualified and candidate) white wine grape varieties (Pallag, 2010(Pallag, -2018


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The Effect of K-Carrageenan Addition to the Characteristics of Jicama Starch-Based Edible Coating and Its Potential Application on The Grapevine

Public awareness of food quality is an essential thing during the packaging application. Considering the environment issue, edible coating with starch-based has been applied as packaging with environment-friendly. The high quality of starch is also produced by jicama (Pachyrhizus erosus). However, the application of jicama as an edible coating for grapevine ( Vitis vinira L) remains unknown. Therefore, this research aims to determine the influence of k-carrageenan addition with various concentration to the physical and chemical characteristics of Jicama ( Pachyrhizus erosus) starch-based edible coating and its potential application on Grapevines (Vitis vinira L.). This research was using randomized design with one addition factor (k-carrageenan): A1 =k-carrageenan 0.5% (w/v); A2 =k-carrageenan 1% (w/v); A3 =k-carrageenan 1.5% (w/v); A4 =k-carrageenan 2% (w/v); A5 =k-carrageenan 2% w/v). All measurements are repeated three times. The collected data were analyzed by variance analysis (p<0.05) and Duncan’s differential analysis for further analysis. The result showed that k-carrageenan addition was significantly influenced the thickness, moisture content, tensile strength value, and WVTR value of the produced edible coating (p<0.05). Treatment A5 has produced films with the good physical characteristics consisting of moisture content at 22.76%, thickness at 1.01 mm, tensile strength at 7.42 kgf/cm2, and WVTR value at 6.748 g/m2 per hour. The edible coating applied on Grapevine with the dipping technique showed that kcarrageenan was significantly increasing the rate of vitamin C and reducing the weight up to day 9 of storage (p<0.05) as compared to control. Meanwhile, based on the sensory evaluation, k-carrageenan addition has insignificantly influenced the color and the texture preference (p > 0.05). Keywords— edible coating; grapevine ( Vitis vinira L.); jicama (Pachyrhizus erosus); k-carrageenan.

I. INTRODUCTION
One of the impacts of the globalization era and population growth is the increase in food demands. This condition is also supported by the improvement of public awareness toward the importance of food quality. Food security as a manifestation of food quality is an important thing to be paid attention, one of which is the packaging application that is easily degraded and does not endanger to the health. For several decades, synthetic packaging such as plastic has been commonly used with the result that caused problems for health and the environment [1], [2]. Packaging technology that is safe and does not damage the environment is very necessary, for example, edible coating [3].
The existence of edible coating application allowed the food packaging can be eaten beside the main function as a coating to inhibit the occurrence of food damage due to the loosing of moisture, oxygen, carbon dioxide, aroma and lipids [4]. During this period, starch which is a polysaccharide has been commonly used as an edible coating because of its ability to reduce the loss of moisture from the food [5]. Because it is biodegradable, starch-based coatings have potency as the plastic polymers replacer [6].
It has been known that beside cassava, the high-quality starch can also be produced from other species of tuber namely jicama. Its scientific name called Pachyrhizus erosus [7]. Previous research reported that the combination of jicama starch with chitosan as an edible coating possessed a good effect on the characteristics of Nile tilapia fish [8]. In that case, chitosan was used as a stabilizer for a suspension. In addition to chitosan, carrageenan is also often used as a stabilizer because it's a hydrophilic characteristic which can increase the continuous phase viscosity so that the emulsion finally stabilizes [9]. Until recent time, the application of jicama starch as an edible coating on fruit, especially grapevine (Vitis vinifera L) has not been done widely. As we know, grapevine is a very perishable fruit with high moisture level so that it is susceptible to evaporation processes which can reduce the quality [10]. Therefore, the application of the edible coating on the grapevine is clearly can play an important role in maintaining its quality.
The current investigation was conducted to get more insight into the effect of carrageenan as the stabilizing agent to the characteristic of jicama starch-based edible coating. Furthermore, the purpose of this study knew of edible coating potency made from a combination of jicama starchcarrageenan was evaluated about its application on the grapevine. The information resulted from this investigation was expected can increase the commodity value of jicama in the field of agroindustry as well as its contribution to the advance of postharvest technology in horticulture commodity.

A. Material
The raw material used in this research consists of jicama (Pachyrhizus erosus), Grapevine (Vitis vinifera L), and kcarrageenan. Reagents used to consist of standard iodine solution (Merck, German) and starch soluble (Merck, German).
B. Research Procedure 1) Extraction of jicama starch: the extraction process was according to Hasbullah (2000) [11] with modification. It was started by peeling and washing jicama. The peeled jicama crushed and added with water (1:1) to produce jicama slurry. The jicama slurry was filtered for paste liquid. Then, it was precipitated for 24 hours and dried at 50°C for 8 hours. Furthermore, it crushed and sieved (80 mesh).
2) Edible film making: Starch was diluted by aquadest with 4% concentration (b/v) using magnetic stirrer and heated on hot plate stirrer at 60-70°C for 20 minutes. Carrageenan was added at various concentrations (b/v) i.e. 0.5 % (A1); 1.0 % (A2); 1.5 % (A3); 2.0 % (A4); and 2.5% (A5) of the total volume in aquadest. Then, the mixture was stirred by using magnetic stirrer and heated on the hot plate up to 80°C. The carrageenan solution homogenized with the starch solution. Furthermore, glycerol 1% added to the solution at 70-80°C. The edible coating solution poured into the 20 x 10 cm glass plate. The, it dried using the oven at 60°C for 5-6 hours.
3) The coating on grapes: The coating process is performed by dipping the fruit into coating liquid. The dipped fruit is then hung and air dried on wire gauze until no drop from the coating. And observation is then performed for 3 days, 6 days, and 9 days at room temperature.

1) Parameters analysis:
The moisture level was analyzed based on the AOAC (1995) [12]. The crushed material sample weighed for 1-2g in a weighing bottle, then dried in an oven at 1000C -1500C for 35 hours depending on the material. After that, put in into exicator and measure the weight. Those treatments were repeated until a constant weight was reached (difference in consecutive weighing is less than 0.2 mg). Weight reduction demonstrated the water content within the material. Coating thickness measured by using micrometer screw gauge (accuracy 0.001 mm). Coating sample inserted between the cylinder spindle and the anvil of micrometer screw gauge. Then, the rated thickness value read by the tool. Tensile strength measured using Llyod Instrument. The tested material cut into the specific shape (under tool specification) and size. The start button pushed twice. The first push would activate the tool and the second push would operate the tool (test in operation). The tensile strength calculated by dividing the maximum force to the coating until it was torn (Newton) by the coating's cross-sectional area (m2). Water vapor transmission rate (WVTR) determined gravimetrically by modifying the method proposed by Xu et al., (2005) [13]. The film sample contained 20 g silica gel, and placed in a container with NaCl solution 40% (b/v) (RH = 75%) at 25oC. The cup has an inner diameter of 75 mm and a height of 30 mm. The water vapor diffused through the film which absorbed by the silica gel and increased additional weight. The cup's weight recorded for 8 hours. A linear regression equation made for the data obtained to determine its slope. The water vapor transmission rate determined by dividing the slope of the cup's weight increment by film's external surface. Weight loss was calculated according to Gardjito (2003) [15]. The deviation of initial weight and final weight (weight during observation) was divided by initial weight resulted in a weight loss of fruit. Vitamin C was calculated by spectrophotometry method. About 200-300 grams of sample was crushed by using blender until it became slurry. Weigh 10-20 grams of slurry and put it into 100 ml measuring flask and added by aquadest. Centrifugation conducted to separate the filtrate or filter it using Gooch crucible to separate the filtrates. Then, filtrates about 5-25 ml inserted into 125 ml Erlenmeyer flask. Add 2 ml starch solution 1% and titrates it using 0.01 N iodine standard solution. Determine the amount of standard iodine and ascorbic acid within the material. 1 ml 0.01 N iodine = 0.88 mg ascorbic acid.

2) Sensory analysis:
The sensory test used is the scoring test by utilizing 20 semi-trained panelists. This test conducted based sensory characteristics such as color, aroma, and texture. The obtained score addressed for statistical analysis to examine the difference. Each coated grape sample was compared based on color and texture with 1 to 5 score. The lowest score demonstrated bad physical characteristics while the highest score showed good physical characteristics.
3) Statistical analysis: The data analyzed by using variance test. Duncan Multiple Range Test (DMRT) at significance level 5% was conducted for further analysis. The parameters such as water content, thickness, tensile strength, and water vapor transmission rate were applied for observation. Moreover, water content, weight loss, vitamin c, and sensory test subjected as further parameters.

III. RESULT AND DISCUSSION
This section presents results and discussion in three main points. It describes the results on edible film water content, water vapor transmission rate, and Grape's water content. The details of this section is presented as follows:

A. Edible Film Water Content
The edible coating's water is content with carrageenan addition with range of 22.76% -27.1%. This result is lower than the edible film using chitosan material as a lactic acid solvent 2% with water content 27.34%-32.48%. Based on variance test results, carrageenan's concentration has noticeable influence (p>0.05) on the coating's water content as presented in Table 1 below.  Table 1 exhibited that there is a difference between A1 treatment with carrageenan's concentration of 0.5% and A5 treatment with carrageenan's concentration of 2.5%. Carrageenan's hydrophilic characteristic induced the binding to the more water which will be evaporated after oven processing. The higher amount of carrageenan on coating will reduce the water content during heating. It makes the coating's texture more rigid than the coating with carrageenan's. The higher the water content on the coating will make the coating vulnerable to fungal growth, which leads to the coating's reduced quality.

1) Thickness:
Based on variance test results, the difference in carrageenan's concentration has noticeable influence (p>0.05) on the coating's thickness. Table 1 demonstrated that there is an increase in thickness in the A1 to A5 treatments. The most significant difference is on the A5 treatment which demonstrated the highest carrageenan's concentration than the other treatments (carrageenan 2.5%). The thickness of the edible coating is influenced by molding area, coating solution volume, and carrageenan's concentration. This is in line with the results of research conducted by Handito (2011). It exhibited that a higher concentration of carrageenan starch causes the increase of edible coating's thickness. Furthermore, it would increase the total dissolved solids existing in the coating forming solution. Thus a thicker film will be produced after the drying process. According to Bourtoom (2007), the coating sheet is formed while the oven process is in progress [6]. Water is evaporated, and the particles are wrinkling to form coating sheet. In brief, the coating forming process starts with reduced space between binding particles in a liquid. Thus a coating sheet will be formed after the evaporation process.
2) Tensile strength: The strength of coating tensile value within range from 0.539 kgf/cm2 (A1) to 7.423 kgf/cm2 (A5). The variance test results show that there is noticeable difference (p>0.05) among coatings A1 (carrageenan 0.5%), A2 (carrageenan 1%), A3 (carrageenan 1.5%), A4 (carrageenan 2%) and A5 (carrageenan 2.5%). The tensile strength values of treatments A1, A2, and A3 are lower than A4 and A5 treatments. The additional concentration of Carrageenan on coating will increase the tensile strength value of edible coating. It assured by the ability of carrageenan to form strong polymer matrix and generated stronger intermolecular tensile strength. This may be viewed on the increasing tensile strength value from A1 to A2 and A3, then to A4 and A5. The A4 and A5 treatments have the highest tensile strength values than the other treatments about 6.240 kgf/cm2 and 7.423 kgf/cm2. The concentration of carrageenan influences the increasing tensile strength value. It exhibited a similar result to the research conducted by Ariska (2015) [15]. The tensile strength value was increased by utilizing coat derived from the core of the banana plant with carrageenan. The higher concentration of carrageenan added into the edible coating to generate a stronger coating matrix. Therefore, it will need a higher force to break the edible coating.

B. Water Vapor Transmission Rate (WVTR)
The WVTR value of coating ranges from 3.198 to 9.603 g/m2/hour. This value is relatively low compared to the results of research conducted by Breemer (2012) [16], in which sweet potato based edible coating with CMC addition has WTVR value ranging from 7.07 g/m2/hour to 12.34 g/m2/hour. Based on variance test results, the concentration of carrageenan noticeably influences the WVTR value of coating. However, there is no WVTR value obtained from A1 treatment which is allegedly caused by low concentration and low thickness of carrageenan. It makes the compactness of coating is not as good as the other treatments. Moreover, the A3 treatment has WVTR value about 3.198 g/m2/hour, which noticeably different from A4 treatment which is 7.256 g/m2/hour. Then, the A2 treatment is 9.603 g/m2/hour which different from A3 treatment and A5 with 6.748 g/m2/hour. The hydrophilic characteristic of carrageenan causes those differences.
According to Handito (2011), more quantity of carrageenan with hydrophilic characteristic in the film matrix increased the film area which may be used to transfer the water vapor [17]. Therefore, the water vapor transmission rate will become higher. Carrageenan is one material within edible coating which classified as hydrocolloid with bad resistance to water vapor. The hydrophilic characteristic of Carrageenan causes the edible coating to absorb water vapor [6] easily.

C. Grape's Water Content
According to Table 2, it showed that on day 3, the water content decreased less than 3% for all treatments. However, on day 6, it is noticeable that in the K treatment, the uncoated grape has the highest decrease at 6.45%. In the A1 to A5 treatments, there is a small amount of water content decreased as compared to the K treatment. Based on the variance test results, it exhibited that there is a difference (p>0.05) in the K treatment (carrageenan 0%) and A5 treatment (carrageenan 2.5%). The A5 treatment is stated as the best since it may inhibit water content for 2.46%. K was 0% of carrageenan's concentration while A1, 2, 3, 4, and 5 were concentration of carrageenan i.e. 0.5; 1.0; 1.5; 2.0; and 2.5% (b/v) respectively. M1-4 was moisture level on day 0, 3, 6, and 9, respectively. Data were presented as mean ± STDEV of 3 repetitions samples (n=3). Different superscripts in the same column showed significant different (p>0.05) On day 9, treatments A1, A2, A3, A4, and A5 may inhibit water content decrease less than 4% except in K treatment which decreased up to 10%. Based on variance test results, there is a more noticeable difference in K treatment (carrageenan 0%) with A5 treatment (carrageenan 2.5%), but there is no difference in all treatments with carrageenan addition (A1, A2, A3, A4, and A5). A1, 2, 3, 4, and 5 were concentration of carrageenan i.e. 0.5; 1.0; 1.5; 2.0; and 2.5% (b/v) respectively. Data were presented as mean ± STDEV of three repetitions samples (n=3). Different superscripts in the same column showed significant different (p>0.05) 1) Grape's weight loss: Grape may experience weight loss along with a longer duration of storage at room temperature. The results of grape's weight loss during storage are given in Table 2. Fig. 1 shows the grape structural without carrageenan and carrageenan added 0,5% and 2,5%. Fig.1 Jicama Starch-Based Edible Coating without k-carrageenan, k-carrageenan 0.5% and k-carrageenan 2.5% to weight losses of grape. The higher concentration of Carrageenan predicted increased the thickness of the coating. Thus, it can cover the space of water molecule which will evaporate from the grape.
On day 3 in Table, 3 showed the weight loss value of grape coated with carrageenan ranges from 2.94% to 4.24%, and the value in K treatment is 12.58%. According to ANOVA test results, there is no noticeable difference (p>0.05) in the weight loss of treatments A1, A2, A3, A4 and A5 during storage. The noticeable difference (p>0.05) occurs in the K treatment, in which uncoated fruit experiences relatively significant to the weight loss at 32.17% on day nine as compared to coated fruit. On day 6 and day 9, the A1 and A5 treatment experienced weight loss differently. In brief, A1 treatment has a weight loss of about 10.64% and 21.35% while A5 treatment has 7.98% and 14.73% of weight loss. K was 0% of carrageenan's concentration while A1, 2, 3, 4, and 5 were concentration of carrageenan i.e. 0.5; 1.0; 1.5; 2.0; and 2.5% (b/v) respectively. W1-4 was weight loss on day 0, 3, 6, and 9, respectively. Data were presented as mean ± STDEV of 3 repetitions samples (n=3). Different superscripts in the same column showed significant different (p>0.05) Weight loss of fruit is caused by respiration activity. Respiration process continues after the harvesting process of fruit. Within the respiration process, oxygen is absorbed to be used in the oxidation process to produce energy, then followed by emission of oxidation remnants in the form of CO2 and water [18].
The weight loss level was getting lower as the concentration of carrageenan increased within each treatment. Inhibition of respiration process is better by increasing the thickness and compactness of coating's molecule binding. The weight loss decreases because of carrageenan's concentration is also found in the research conducted by [19].
2) Vitamin C content of grape: Generally, fruit experiences oxidation process within the storage. This process is decreasing the vitamin c content of grape. Vitamin C is easily oxidized to L-dehydroascorbic acid, and it tends to experience a further change to L-diketogulonic [20]. The results of vitamin c test during storage are presented in Table  4. The vitamin c content on day 0 and day 3 is within range 4.033 mg/100g to 4.308 mg/100g and 3.942 mg/100g to 4.033 mg/100g, respectively. Based on the variance test results, the concentration of carrageenan does not noticeably influence (p>0.05) vitamin c content on day 0 and day 3. The difference (p>0.05) has seen on day 6 in K treatment (carrageenan 0%), A1 treatment (carrageenan 0.5%), and A5 treatment (carrageenan 2.5%). High concentration of carrageenan in the A5 treatment generates strong coating sheet to inhibit the oxidation process at room temperature.
The similar result is found on day 9, the concentration of carrageenan significantly influencing all treatments. The A1 treatment has the least vitamin c content as compared to the other coatings with 2.842 mg/100g grape. While A2, A3, A4 and A5 treatments may maintain the vitamin c content up to ≥3.483 mg/100g grape.
3) The sensory characteristic of grape: The evaluation of fruit quality involves the sense of taste of compounds which influence the aroma, flavor, and crispiness of fruit. By determining the quality of those critical components, then it must be combined with the panel's subjective evaluation to provide meaningful information on fruit's quality [21], [22]. The results of the grape's sensory test during storage are given in Table 5. The color tests of treatments A1, A2, and A3 have an average value higher than A4, and A5 treatment since the coating's color of fruit within A1, A2, and A3 treatment is clear. Although there is no difference within each treatment, the concentration of carrageenan within coating influences its color. Grapes with A4 and A5 treatment have a clear yellow color which tends to be white because of the relatively thick coating.
In the grape's surface texture test, there is no skin wrinkling within each treatment. The reduction of weight loss and water content will make the texture of fruit turn into wrinkled. The texture test value of each treatment is not significantly different since the external layer of fruits within all treatments is sturdy texture According to the ANOVA test results, there is a difference (p>0.05) in the aroma value of A1, A2, and A5 treatments. The A1 and A2 treatment has low values of grape's aroma such as 2.62 and 2.57, respectively. Meanwhile, the A5 treatment is the highest value of grape's aroma about 3.71 since the sour aroma of grape in A5 treatment is not strong compared to the other treatments.

IV. CONCLUSION
This study concludes that the k-carrageenan 2.5% may become the best-selected treatment for grape edible coating application with increasing vitamin C rate and lowest weight lost level. However, based on the sensory treatment, kcarrageenan as the best treatment as the grape coating has a pure color, soft texture, and no acidic color.


extfieldsofstudy: Biology Medicine

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Association of Tomato Leaf Curl New Delhi Virus, Betasatellite, and Alphasatellite with Mosaic Disease of Spine Gourd (Momordica dioica Roxb. Willd) in India

Background Spine gourd (Momordica dioica Roxb. Willd) is one of the important cucurbitaceous crops grown across the world for vegetable and medicinal purposes. Diseases caused by the DNA viruses are becoming the limiting factors for the production of spine gourd reducing its potential yield. For the commercial cultivation of the spine gourd, propagation material used by most of the growers is tuberous roots and stem cuttings, which in turn results in an increased occurrence of the mosaic disease. There is a need for understanding the causal agent; through characterization of which will lead to the designing management strategies for the spine gourd mosaic disease control. Objectives Characterization of a begomovirus and its satellites associated with mosaic disease on spine gourd. Materials and Methods Total DNA was extracted from spine gourd samples exhibiting symptoms typical to the begomoviruses infection (mosaic mottling, leaf curl) and was tested by PCR using begomovirus specific primers. Furthermore, the complete genome of begomo viruses (DNA A, DNA B, alpha satellite, and beta satellite) was amplified by rolling circle amplification (RCA) method. Results The full-length sequences of DNA A, DNA B, alpha satellite, and beta satellite isolated from symptomatic spine gourd were determined. The full length genomes (DNA A and DNA B) of the Tomato leaf curl New Delhi Virus (ToLCNDV) infecting spine gourd were compared with the other begomovirus genomes available in the data base. The sequence analysis has revealed that DNA A and DNA B components of the begomovirus infecting spine gourd share 95.4–96.2 and 86.7–91.2% identical sequence (i.e., nucleotide (nt) identity) with that of ToLCNDV infecting potato and cucurbits in the Indian subcontinent isolates reported earlier (available in GenBank), respectively. Further, alpha satellite and beta satellite were also detected in the begomovirus infected spine gourd samples. The recombination analysis of the DNA A, DNA B, beta satellite, and alpha satellite of the begomovirus infecting spine gourd showed the associated begomovirus and satellite DNAs were driven from the different begomoviruses, leading to emergence as a new variant of the begomovirus infecting spine gourd. Conclusions The commercial cultivation of the spine gourd by most growers depends on the tuberous roots and stem cutting. The occurrence of begomovirus in spine gourd gives an alarming signal against utilization of such infected plant materials in the crop breeding and improvement programs. Using the clean virus-free vegetative propagation material is considered as one of the most important methods for controlling viral diseases. The study is highly useful for detection of the begomovirus infecting spine gourd in the detection of the virus infection in the clonally propagated planting material.

Background
Spine gourd (Momordica dioica Roxb. Willd) is a perennial, rhizomatous, dioecious, climbing creeper which belongs to the family of Cucurbitaceae and commonly is known as Kakrol. It is originated from Indo-Malayan region and distributed throughout India, China, Nepal, Bangladesh, Myanmar, Pakistan, and Sri Lanka (1). In India, the crop is being widely cultivated in Orissa, Maharashtra, Karnataka, Andhra Pradesh, Bihar, and West Bengal for its good taste and high nutritional value. Generally, spine gourd is largely cultivated through vegetative propagation and is less propagated by seeds (2). The edible fruits contain a high amount of protein, carbohydrate, fiber, moisture, ash, iron, calcium, phosphorus, thiamine, riboflavin, and niacin (3). Apart from nutritional value, all parts of spine gourd have medicinal properties to cure various diseases and disorders in human being. It has a huge demand in the market, but still remains underutilized (4,5) and commercially under-exploited due to its vegetative mode of propagation, dioecious nature, low percentage of the seed germination, and a long period of seed dormancy (4). The commercial propagation of the spine gourd mainly depends on the tuberous roots followed by stem cuttings. The Spine gourd plants showing symptoms typical to the begomoviruses' infection in the cucurbits is emerging as the major constraint for its production and making the availability of the healthy planting material difficult in the country. Begomoviruses are single-stranded DNA viruses belonging to the family Geminiviridae with the morphology of geminate particles and are transmitted by the whitefly (Bemisia tabaci). Begomovirus could either have a monopartite or bipartite genome. The bipartite begomoviruses have two genome components referred to as DNA A and DNA B. The DNA A component contains five open reading frames (ORFs) encoding pre-coat protein, coat protein in the virion strand, as well as DNA replication-associated proteins in the complementary strand (6). The DNA B component contains two ORFs and encodes factors required for inter and intra-cellular movement in the host plants (7). Furthermore, based on the genome organization, genetic diversity, and geographical distribution, it has been further divided into two groups; the old world (OW) (Europe, Africa, Asia and Australia) and the new world (NW) (America) begomoviruses. The NW begomovirus is evaluated to be bipartite with lack of AV2 ORF in the DNA A component. Whereas both bipartite and monopartite begomovirus in the OW encodes AV2 ORF. The monopartite begomoviruses have a single genome analogous to the DNA A of the bipartite viruses with the association of additional ssDNA molecules known as betasatellites and/or alphasatellites (DNA1) (8). Betasatellites are approximately half the genome size of their helper begomoviruses and are required for inducing typical disease symptoms in their original hosts (9). Alphasatellites are self-replicating circular ssDNA molecules and depend on the helper virus for movement, encapsidation, vector transmission, and play no role in symptom induction (10).

Objective
Spine gourd is consumed by tribal groups living around the natural forest areas, especially at higher altitudes, where the native folks consume it as a daily vegetable. The spine gourd did not gain much popularity until it was discovered to have a high nutritional and medicinal value, which helps the development of body towards natural immunity from many common ailments. The disease caused by the begomovirus is the major constraint for production of the healthy planting material as well as its production in the country. The virus infected plants are exhibiting severe mosaic, mottling, and leaf curl symptoms. The disease incidence is ranged from 50-60% across different farmer's fields at Varanasi, Uttar Pradesh state of India. Therefore, the current study was undertaken to characterize the probable begomovirus and its associated satellites with the mosaic disease of the spine gourd in India.

Virus Source
Five leaf samples of spine gourd exhibiting mosaic, mottling, leaf curl, and distortion symptoms ( Fig. 1A-1D) and two samples from non-symptomatic plants were collected during 2014-15 from different farmer's fields of the Varanasi, Uttar Pradesh state, India.

DNA Isolation, PCR-Mediated Amplification, and Sequencing
Total DNA was extracted from symptomatic and nonsymptomatic leaf samples using CTAB (11). The presence of begomovirus infection in the spine gourd was tested by PCR using begomoviruses specific degenerative primers (12). The full-length genomic DNA components were amplified from the virusinfected samples using a TempliPhi illustra amplification kit (GE Healthcare, Piscataway, NJ). The resulted rolling circle amplification (RCA) product was digested with BamH1 (DNA-A) and XbaI (DNA-B) restriction endonucleases for isolation of the monomeric units of the genome, cloned into pUC19 plasmid (13), and were transformed into the competent Escherichia coli DH5α strain. Restriction digestion was performed for confirming recombinant clones. Similarly, to identify the association of the sample with satellite genomes, the total DNA was amplified using betasatellite (DNA-β) (14) and alphasatellite (15)

Sequence Analysis and Detection of Recombination Events
The sequences obtained were initially analyzed using the Vector NTI AdvanceTM 9 software to remove vector sequences. Further, the sequences were verified for the presence of all begomovirus specific Open reading frames (ORFs) using NCBI ORF finder and conserved nonanucleotide sequence. The selected begomovirus species (Table S1), betasatellites (Table  S2) and alphasatellites (Table S3) accessions showing highest percentage sequence identity/similarity/ homology with the current sequences in the study were retrieved from the GenBank for analysis. The retrieved sequences were aligned with the present isolate using the MUSCLE method in SDT version 1.2 (16) and percent pair-wise nt identities were generated. The phylogenetic tree was generated by MEGA 7 software (17) using the neighbour-joining method with 1,000 bootstrapped replications to estimate evolutionary distances between all pairs of the sequences. Splitdecomposition trees were constructed with 1,000 bootstrap replicates based on parsimony splits as implemented in SplitsTree version 4.11.3 with default settings (18). Recombination analysis was carried out using the recombination detection program (RDP), GENECOV, Bootscan, Max Chi, Chimera, Si Scan, 3Seq which are integrated in the RDP4 (19). The default RDP settings with 0.05 P-value cut off throughout and standard Bonferroni correction were used.

Genome Organization of DNA A Component of Begomovirus
The spine gourd leaf samples showing symptoms typical to the begomovirus infection were showed positive PCR amplification to begomovirus specific primers and no amplification was observed from non-symptomatic samples. Further, the full genome components (DNA A and DNA B) were amplified and the resultant amplified products of 2.7 kb were cloned into pUC19 plasmid. The representative ten recombinant plasmids were sequenced and confirmed by BLAST analysis. The DNA A sequences of the five begomovirus isolates characterized from spine gourd showed 99.8-100 percent nt sequence identity among themselves indicating that they belong to a single species, based on species demarcation criteria for the begomoviruses; 91% nt sequence identity for complete genome (20). Therefore, one representative from the begomovirus isolate of SPYG1 was selected for amplification of the DNA A and DNA B as well as alpha and betasatellite of the begomovirus infecting spine gourd.

Sequence Identities of the DNA A and DNA B Components with Other Begomoviruses
The  Table 2). Based on the current species demarcation criteria for the begomoviruses (91% nt sequence identity among complete genome) (20), the begomovirus isolate (SPYG1) with more than 95 percent nt sequence identity with ToLCNDV-pot is considered as a variant of the ToLCNDV. This result was well supported by the phylogenetic analyses of the both DNA A and DNA B components of the SPYG1 isolate by grouping with Indian isolates of ToLCNDV infecting tomato, potato, and cucurbits crops in the India and China ( Fig. 2A and 2B).

Genome Organization of the Alpha and Betasatellite and Sequence Affinities to Other Alpha and Betasatellite
The alphasatellite (DNA D1) and betasatellite (DNA β1) isolated from infected spine gourd were determined to be 1388 nt (Acc NO. MG571523) and 1360 nt (Acc. NO. MG571522) in length, respectively. The alphasatellite have a single large ORF with a coding capacity of 315 aa in the virion-sense (coordinates 89-1036) (21). The sequence showed the maximum nt sequence identity (76.9%) with Tomato leaf curl Karnataka alphasatellite (ToLCKVD1, Acc. NO JX570736) ( Table 4). Since the alphasatellite sequence was showing very low levels of nt sequence identity (less than 77%) with other isolates reported earlier and there is no proposed species demarcation threshold for classification of the alphasatellites, the present alphasatellite will be named as spine gourd mosaic alphasatellite. The phylogenetic analysis of this studied genome component shown in Figure 3A indicated that the alphasatellite isolated from spine gourd segregated separately from all other satellites infecting different crops. Similarly, betasatellite sequence in the current study contains all the features typical to the betasatellites reported so far (12,14) and showed maximum nt identity of 81.7 to 87 percent with the tomato leaf curl Jodeypur betasatellite (ToLCJoB) isolates originating from the Indian subcontinent infecting different crops ( Table 3). Based on the recently proposed species demarcation threshold for betasatellites (22), the betasatellite identified here is an isolate of ToLCJoB infecting chilli and tomato, which is well supported by phylogenetic analysis (Fig. 3B).

Neighbor-Net and Recombination Analysis
A neighbor-network, Pairwise Homoplasy Index (PHI) test and breakpoint analysis for the recombination was carried out for nt sequences of the different begomoviruses genomic components (DNA A, DNA B, betasatellite, and alphasatellite) along with the sequences of the virus isolated from the spine gourd. This revealed that SPYG1 isolate was evolved through recombination. The RDP 4 analysis indicated the evidence of recombination in ToLCDNV (SPYG1) infecting spine gourd with most of the DNA A fragments derived from ToLCNDV, Squash leaf curl China virus (SLCCNV) and Tomato leaf curl Palampur virus (ToLCPaV) to emerge as a new strain of ToLCNDV infecting spine gourd (Table S4, Fig. 4).  In case of DNA B sequence, most part of BV1 (movement protein) and BC1 (nuclear shuttle protein) is derived from ToLCNDV and ToLCPaV infecting tomato, okra, and melons.The IR region is derived from the Mungbean yellow mosaic India virus (MYMIV) and ToLCNDV (Table S4, Fig. 4). The RDP analysis indicated that evidence of recombination in betasatellite suggestive of the most parts of betastallite DNA descended from Tobacco leaf curl Patna betasatellite (ToLCPnB), Tomato leaf curl Bangladesh betasatellite (ToLCBDB), and Tomato leaf curl Joydebpur betasatellite (ToLCJoB) infecting different solanaceous, and malvaceous crops in India. The analysis of the alpha-satellite indicated that most of satellites DNA descended from Cotton leaf curl alphasatellite (CLCuVD1), Cyamopsis tetragonoloba leaf curl alphasatellite (CyTLCuD1), Okra leaf curl alphasatellite (OLCuDD1), and Sida leaf curl alphasatellite (SiLCNVD1) satellites (Table S3) are associated with cotton, guar, okra, and tomato infecting begomoviruses, respectively.

Discussion
Spine gourd is one of the cucurbitaceous crops and mosaic disease associated by begomoviruses is a limiting factor for its production. The ToLCNDV was initially identified on solanaceous crops in India (23), since then it has been reported across many countries and known to infect 43 plant species belong to the different families (24)(25)(26). The frequency of the newly emerging strains of the ToLCNDV on several cultivated and non-cultivated plant species are increasing in the recent years, indicating that the virus poses a serious threat to vegetables belonging to the family Cucurbitaceae, Euphorbiaceae, Solanaceae, Malvaceae and Fabaceae throughout the world (26). Generally, the betasatellites are found associated with the old world (OW) monopartite begomoviruses (10) and not in bipartite begomoviruses with the two exceptions, MYMIV (27,28) and ToLCNDV (29) infecting mung bean and tomato, respectively in India. In the present study, for the first the time, we have characterized ToLCNDV bipartite virus with both betasatellite and alphasatellite associated with the mosaic disease of the spine gourd in India. The tripartite interaction between ToLCNDV and betasatellite in tomato was well proved through agro-inoculation of the cloned DNA A, DNA B, and betasatellites in the N. benthamiana and tomato (30). It was evident that the plants co-inoculated with DNA A, DNA B, and betasatellites will enhance the symptom severity in the N. benthamiana and tomato directly increases the helper viral DNA A and DNA B replication levels in the host cells. The betasatellite associated with spine gourd mosaic disease is identified as ToLCJoB, which is also associated with the heterologous begomoviruses infecting chilli (31,32), tomato (33), and kenaf (34) plants in India. The trans-replication of ToLCNDV and defective betasatellites has been shown for many distinct begomovirus species/betasatellite combinations (30,(34)(35)(36). The other bipartite begomoviruses confirmed to trans-replicate betasatellite are Sri Lankan cassava mosaic virus (36), Tomato yellow leaf curl Thailand virus (37), and the New World bipartite Cabbage leaf curl begomovirus (38). These observations clearly indicated that there is no distinct demarcation between monopartite and bipartite begomoviruses for their ability to trans-replicate betasatellites in the genome. The nt sequence of alphasatellite isolated from spine gourd showed less than 77 per cent identity with the previously reported alphasatellites, hence we proposed the name spine gourd mosaic alphasatellite. The exact role of alphasatellites has not been fully understood.
However, alphasatellites attenuate the disease symptoms and involve in the maintenance of the low level of betasatellite accumulation (39). It was also shown that the Rep of alphasatellites can suppress RNAi pathway in monopartite begomovirus disease complexes (40). The PHI value in the present study has strongly supported the presence of recombination in SPYG1 isolate. The different methods used for recombination breakpoint analysis provided strong evidence for the presence of the past recombination events in most of the genome components. The role of such overlapping recombination between different isolates or different species in adaptation to spine gourd may be an interesting aspect that needs to be resolved. Such inter and intra-species recombinations are the predominant feature of begomovirus evolution (8) and have been implicated in the emergence of new begomovirus species and adaptation in new hosts in the agricultural system (40). The commercial cultivation of spine gourd by most of the growers is depends on the tuberous roots and stem cutting. The occurrence of the mosaic disease on spine gourd gives an alarming signal against utilization of such virus infected planting materials in the crop breeding and improvement program. The technique developed here will be highly useful to detect the virus infection in clonally propagated plants such as spine gourd.