Application of microwave treatment for reduction of microbial load in jaggery cubes

Abstract

In present study, fresh jaggery cubes were used and treated with microwave heating. Two treatment time has been selected to treat the jaggery such as 30 s and 45 s. Physiochemical and microbial parameters were analysed before and after treatment of microwave. Moisture content of treated jaggery sample was 3.98 ± 0.59 and 3.86 ± 0.63 for 30 s and 45 s microwave treatment respectively. Similarly, this treatment maintains the water activity at 0.6, which is considered as a safe for consumption. The content of iron retains in this microwave treatment; it doesn’t affect much on the iron content present in fresh Jaggery sample. Most substantial result has been observed in case of total plate count and yeast and mold count when treated with microwave heating. In this case microwave process at 45 s was found to be the best since it reduced the total plate count (TPC) load to 13 ± 4 from 194 ± 48 cfu/g. Also, in case on yeast and mold count, 45 s microwave treatment showed reduction of load to 28 ± 7 cfu/g from 25,498 ± 2809 cfu/g. Present investigation showed that microwave treatment for 45 s was found to be the best to treat the jaggery.

Introduction

A natural, conventional sweetener created by concentrating sugarcane juice is known as jaggery (also known as gur). Traditional consumers of this unprocessed, non-centrifugal sugar are found in Asia, Africa, Latin America, and the Caribbean. It is recognized as the healthiest sugar on the planet since it keeps all the vitamins and minerals contained in sugarcane juice (R. Kumar and Kumar 2018). Its colour ranges from golden brown to dark brown and it is a concentrated cane juice product without the molasses and crystals being separated. India is the world's top producer and consumer of jaggery. More than 70% of the world's entire production comes from India (Rao et al. 2007). It is consumed as a sweetener and as a food in practically all societal segments. It is also consumed in animal feeds. In Ayurveda, it is used as medicine, blood purifier and basic component for syrups (D. Kumar et al. 2013).

Jaggery is manufactured between October and April but since it is marketed and consumed throughout the year a substantial part of it has to be stored. It has been estimated that one third to one half of the jaggery produced every year is stored for consumption during and after rainy season. Jaggery contain hygroscopic substances such as reducing sugars, dextrose, minerals like chlorides, sodium, potassium which make the jaggery liable for moisture absorption and microbial degradation. The estimates showed that 5–10% of stored jaggery is lost because of its hygroscopic nature. It is difficult to store jaggery during the periods of high humidity and high temperature. These conditions fall during monsoon season (July to September) when the relative humidity values exceed more than 55–60% due to which it may lose its normal texture, colour and flavour microbial growth causing compositional changes leading to undesirable aroma, flavour and quality deterioration. Jaggery is considered as a traditional Indian sweetener and has been consumed by most of the people as a part of food ingredients in various sweet, traditional as well as savory dishes. Due to its taste, colour and nutritional aspects has a huge demand in the market, hence jaggery has been produced in different size and shapes such as Ladoo (bheli—small piece of jaggery in the form of ball having weight 50 to 250 g), Dhayya or bheli (big round piece having weight around 1 to 2 kg), Pari (semi round pieces of 1 to 2 g), Chaukunta/Chausera (4 to 5 kg pieces in squared shape), Pansera (semi round pieces of 5 to 6 kg), Dhansera (semi round pieces of 9 to 10 kg), Balti (bucket shape jaggery blocks having size of 10–20 kg), Basket (Jaggery moulded using bamboo basket having weight around 10 to 15 kg), Chaku (irregular broken jaggery blocks of size varying from 1/2 to 3 kg), Khurpapad (square pieces of 250–300 g). Production of jaggery in such different shape and size results in poor handling, improper packaging, and difficult for distribution and transportation (Anwar 2017; Anwar et al. 2015; Singh et al. 2011). To standardize the size and shape of jaggery, Indian Institute of Sugarcane Research (IISR), Lucknow developed jaggery moulding frame for moulding jaggery in 500 g bricks (Baboo et al. 1988). Further it has been modified to 25 mm cube shape and is very much liked by the people and the frame for such shape is in much demand (Anwar 2017). In present study we have used cubical shape moulds having size (25 mm × 26 mm × 17 mm) for easy to handle and packaging.

Microwave heating is based on the transformation of alternating electromagnetic field energy into thermal energy by affecting polar molecules of material microwave treatment has been widely used for the agricultural products to reduce their moisture content as well to enhance their keeping quality. Microwave treatment is considered more suitable because of its low operational cost and more precise control during heating as compared to other heat-treatment methods (Orsat, Raghavan and Krishnaswamy 2017; Orsat et al. 2007). It is a continuous process that allows large quantities of products to pass in a shorter period of time with no chemical residues left in the food and hence no adverse effects on human beings. The major problem associated with jaggery storage is the presence of invert sugars and mineral salts which being hygroscopic in nature absorbs moisture particularly during monsoon season when ambient humidity is high and lead to spoilage. During storage, jaggery basically suffers from four types of deterioration: physical, chemical, biological and microbiological. The main problems related to solid jaggery storage are running-off (liquefaction) and deterioration of color during storage (Chand et al. 2011). These problems arise due to quick absorption of moisture and microbial attack on jaggery. Jaggery from mature cane recorded less reduction in quality parameters under cold storage compared to the jaggery from immature and over aged cane. An effective way to control this excess moisture development inside the package is to use a moisture absorber. Moisture absorber regulates water activity of the product to suppress the microbial growth (Choi & Lee 2013). Therefore, the aim of these study was to evaluate the effect of microwave heating on reduction of microbial load of jaggery.

This method of preservation is based on two principles: dipole interaction and ionic conduction. Normal jaggery has 6 to 7 percent moisture, which is made up of polar molecules like water. The orientation of these molecules is usually unpredictable. When an electric field is introduced, however, the molecules orient themselves in accordance with the field's polarity. When jaggery is exposed to microwave radiation over a brief period of time, the polarity shifts very quickly, for example, at a microwave frequency of 2,450 MHz, the polarity shifts 2.45 billion cycles per second. This rotation of molecules causes friction with the surrounding media, resulting in the generation of heat, which kills the microbes present in jaggery. Because jaggery includes moisture in the range of 3 to 9%, when it is exposed to microwave radiation, the ions travel at a faster rate due to their inherent charge. The resultant collisions between the ions result in the conversion of the moving ions' kinetic energy into thermal energy, resulting in the killing of microbes at lower temperatures (less than 50 °C) and in a short period of time. Thus, our goal is to investigate the impact of microwave treatment, a low-temperature advanced food processing approach that can reduce microbial load without compromising the nutritional content of jaggery according to the above concept.

Materials and method

Materials

Solid jaggery cubes (weight = 14 ± 1 g with dimension 25 mm × 26 mm × 17 mm) were kindly gifted by the Paawak Foods for the research purpose. Moisture absorbers and oxygen scavengers were procured from the Sorbead India, Gujarat, India.

Methods

Microwave treatment

To understand the changes and mechanism of microwave treatment on jaggery cubes, the obtained jaggery samples were kept at 55 °C for different time cycle. A domestic microwave oven (LG, Model MS2043BP) having frequency of 2450 MHz, power output varies between 100 to 700 W with dimension (455 mm × 252 mm × 320 mm) was used for the present experiment. The time–temperature cycle used in this experiment were 50 °C for 30 s, and 50 °C for 45 s. Jaggery cubes without Microwave heating and with microwave heating at different time cycles were compared and suitable time–temperature cycle was optimized to improve the keeping quality of jaggery.

Moisture content and water activity analysis

Moisture content in all samples was assessed gravimetrically. For this, 2 g of jaggery was placed in an aluminum pan, and dried in a hot air oven at 105 °C for 3 h. Moisture content was calculated using Eq. (1).

$$\text{Moisture}\left(\text{wet basis}\right)\text{\%}=\frac{\text{Initial weight of sample}-\text{final weight of sample}}{\text{Initial wieght of sample}}\text{X}100$$ 1

Water activity (a_w) was determined using a water activity meter (Aqua Lab dew point, 4TE Decagon Devices Inc., USA). Samples were kept in sample holders and a_w was measured at the room temperature.

Estimation of iron and vitamin C in jaggery sample

The iron content on the jaggery sample was estimated by the method given in the FSSAI manual (FSSAI 2016). For determination of vitamin C in jaggery sample the method described by FSSAI has been followed (FSSAI 2019). Initially 50 mL of jaggery solution is pipette out into a 100 mL volumetric flask, then 25 mL of 20% metaphosphoric acid has been added as stabilizing agent and diluted it. Further 10 mL of prepared solution was pipette out in a small flask and 2.5 mL acetone has been added to it. Followed by titration with indophenol solution until a faint pink colour persists for 15 s.

$$\text{Vitamin C}\left(\text{mg}/100\text{mL jaggery solution}\right)=20\left(\text{V}\right)\left(\text{C}\right)$$ 2

where,

V = mL indophenols solution,

C = Vitamin C per mL indophenol solution.

Antioxidant activity of jaggery

Antioxidant activity of the jaggery solution was evaluated by the free radical scavenging activity of the solution against 2, 2-diphenyl-2-picrylhydrazyl (DPPH) radical. The DPPH antioxidant assay was carried out according to a method described by Nayaka et. al., with some modifications (Nayaka et al. 2009).

The working DPPH solution was prepared by diluting stock DPPH solution (0.24 g/L) with methanol to obtain an absorbance of 1.1 ± 0.02 at 515 nm. A volume of 0.15 mL of jaggery solution then mixed with 2.85 mL of the DPPH working solution in a test tube and the solution was left for 15 min at room temperature in a dark room. The absorbance of the reacted solution was then measured at 515 nm using a spectrophotometer (V-630, Jasco, Japan). The absorbance of a blank sample that contained the same amount of methanol and DPPH solution was also measured.

Radical scavenging activity was calculated by the following formula:

$$\%\text{Inhibition}=\left[\left({\text{A}}_{\text{b}}-{\text{A}}_{\text{e}}\right)/{\text{A}}_{\text{b}}\right]\times 100$$ 3

where A_b is the absorbance of the test with a blank sample and A_e is the absorbance of the test with jaggery solution. Then % of inhibition was plotted against concentration, and from the graph IC50 was calculated. The experiment was repeated three times at each concentration.

Microbial analysis

The microbial tests were carried out as per the IS standard; Briefly, in sterile conditions, 1 mL of 10⁻¹, 10⁻² and 10⁻³ dilutions of 10 g of jaggery and 90 mL of buffer solution of KH₂PO₄ (pH 7.0) previously homogenized using magnetic stirring at 125 rpm were cultured in petri dishes using Tryptone Yeast Extract Agar medium (Sigma-Aldrich, Merck). Once the culture medium was solidified, the petri dishes were incubated inverted for 24 h at 35 °C to obtain the mesophilic aerobic bacteria colonies count.

For the yeast and mold counts, 1 mL dilutions of the samples were placed in petri dishes, with 15 mL of potato-dextrose agar culture medium (Sigma-Aldrich, Merck). The dishes were incubated at 22 °C for 48 h and the colonies count was carried out considering the dilution factors. For the total coliform count, 1 mL dilutions in phosphate buffer were inoculated in petri dishes containing Violet Red Bile Lactose Agar medium (Sigma-Aldrich, Merck). The petri dishes, in an inverted position, were incubated at 35 °C for 24 ± 2 h. Microbial analyses were done on samples of treated as well as untreated jaggery in the laboratory. The microbial counts were done every months for period of 9 months (Solís-Fuentes., J. A & M, 2019).

Statistical analysis

All experiments were conducted in triplicate, with three independent sample sets. The data were represented as mean ± standard deviation (SD). To evaluate significant difference among the means, one-way analysis of variance (ANOVA) was performed at a significance level p ≤ 0.05 using SPSS 20.0 (IBM, Armonk, NY, USA).

Result and discussion

Moisture content

Moisture content is an important parameter which favors growth of microorganism and is responsible for spoilage of food. In the present study, effect of microwave treatment as a probable method for preservation of jaggery is investigated. The jaggery was given microwave treatment for 30 s and 45 s and it was compared with the control sample. The results are shown in Table 1. It is observed that microwave treatment positively reduces moisture content without effecting quality of jaggery. It was noticed that microwave treatment resulted in reduction of moisture content from 4.41 ± 0.726 to 3.98 ± 0.19 and 3.86 ± 0.23% when treated at 30 s and 45 s respectively (Fig. 1). It is observed that with increase in the treatment time there was decrease in the moisture content of jaggery. The moisture content of control sample was higher as compared with the treated samples of jaggery. Microwave energy is transmitted to the molecules at the molecular level. The electromagnetic waves interact with the molecules in food material by molecular friction resulting from dipole rotation of water molecule present in the food material. Food absorbs microwave energy during microwave treatment, which generates heat in food material. This results in increase in the temperature, pressure and also the concentration gradients increase which results in removal of moisture because of increase in the flow rate of moisture from food to surface. Hence, in present work it is observed that as the microwave exposure time increases the flow rate / removal of moisture from jaggery also increases without any significant effect on the texture of jaggery. So, microwave treatment of jaggery for a optimum time can reduce the moisture content of jaggery and could help in increasing shelf-life of jaggery cubes. Carvalho et al., (Carvalho et al. 2021) studied effect of microwave on the moisture removal of barley malt. The authors reported that the microwave treatment to barley malt resulted in increased rate of removal of moisture in shorter time as compared with the traditional process. So, microwave treatment of jaggery result in reduction of moisture content and thus enhancing the shelf-life of jaggery.

Table 1.

Effect of microwave treatment on physiochemical and microbial parameters (T1: Microwave heating at 30 s T2: 45 s)

Parameters	Control	T1	T2
Moisture	4.41 ± 0.726b	3.98 ± 0.19a	3.86 ± 0.23a
Water Activity	0.6131 ± 0.001c	0.6075 ± 0.005b	0.6048 ± 0.003a
Iron	9.028 ± 0.77a	8.817 ± 0.46a	8.382 ± 0.67a
pH	5.216 ± 0.22a	5.23 ± 0.24a	5.698 ± 0.21b
Vitamin C	4.464 ± 0.21b	4.39 ± 0.14a	4.747 ± 0.24b
Antioxidant Activity	32.73 ± 0.33a	32.67 ± 0.29a	32.47 ± 0.3a
Total Plate Count (cfu/g)	194 ± 48c	87 ± 14b	13 ± 4a
Yeast and Mold (cfu/g)	25,498 ± 2809c	79 ± 12b	28 ± 7a

Fig. 1.

Effect of microwave treatment on moisture content and water activity of jaggery samples (T1: 30 s treatment; T2: 45 s treatment)

Water activity

Water activity is important parameter which not only control the microbe’s growth but it also has an impact on the various biochemical and chemical reactions which occurs in food material. The values of water activity obtained in present work are given in Table 1. It was observed that the water activity of samples treated for 30 s and 45 s was 0.6075 ± 0.005 and 0.6048 ± 0.003 respectively. It is seen that with increase in the treatment time there is marginal decrease in the water activity in jaggery cubes. But the untreated sample/ control sample showed higher water activity of 0.6131 ± 0.001, which was much higher than those reported in microwave treatment (Fig. 1). To prevent spoilage and to enhance the shelf-life of food it is important to estimate the water activity. Most of the microorganisms grow well in optimum water activity leading to spoilage of food. Generally, water activity of more than 0.85, results in quick spoilage of food and food need to be refrigerated. But, water activity below 0.85 and more than 0.60 also results in spoilage but comparatively at slower rate and water activity below 0.60 shows higher shelf-life without refrigeration (Serin et al. 2018). In present work water activity of control jaggery sample was more than 0.60, whereas microwave treatment significantly reduced the water activity to 0.6075 ± 0.005 and 0.6048 ± 0.003, indicating that microwave has beneficial role in reducing water activity and increasing shelf-life of jaggery because all microbial growth is arrested at water activity below 0.60. Hence, the treatment time of 45 s was the optimum for reduction in water activity in jaggery cubes, which enhanced the shelf-life of cubes stored in ambient temperature without refrigeration.

Iron and vitamin C content

Jaggery is known for its mineral content and the beneficial effect of jaggery are because of this mineral content. One such very important mineral is Iron, which is required for normal growth and development of human body. In present research the effect of microwave treatment on the iron content of jaggery is checked and obtained results are summarized in Table 1. The Iron content of jaggery decreases with the increase in microwave irradiation exposure time. The Iron content of jaggery sample treated for 30 s and 45 s was 8.817 ± 0.46 and 8.382 ± 0.67 respectively (Fig. 2). The Iron content of untreated sample was 9.028 ± 0.77, which was higher than the treated samples. Though, there was decrease in the Iron content with increase in the irradiation time but it was within the acceptable limits and was not much significant as compared with the untreated sample. Unlike Iron content, there was changes in the content of Vitamin C in treated sample as seen from Table 1. The vitamin C content in jaggery samples treated for 30 s and 45 s was 4.39 ± 0.14 and 4.747 ± 0.24 respectively whereas untreated sample showed vitamin C content lower than the sample treated for 45 s. There was significant difference in the vitamin C content of untreated and treated sample, the vitamin C content of treated sample was much higher than untreated sample. Usually, microwave treatment has little or no effect on the mineral and vitamin content of food upto optimum level of treatment (Jiang et al. 2017). In the present study the optimum exposure time of 45 s was found to be beneficial in terms of vitamin C content though at 45 s the iron content was marginally lower. Brown et al., (Brown et al. 2020) studied comparison between traditional heating and microwave heating of food. The authors reported that the food constituents including protein, fat, carbohydrates and minerals are retained in sample treated with microwave. The authors further added that the traditional heating and microwave irradiation showed no significant differences in nutritional content of food except for vitamin C. The vitamin C content of microwave irradiated samples was much higher as compared with the traditionally heated food. Hence, the results obtained in the present work are in line with the results reported in the literature. The microwave irradiation is probably an excellent method for preservation of jaggery without compromising the nutritional content of jaggery cubes.

Fig. 2.

Effect of microwave treatment on iron content, Vitamin C and pH of jaggery samples (T1: 30 s treatment; T2: 45 s treatment)

Antioxidant activity

The antioxidant potential of treated and control jaggery samples is summarized in Table 1. As seen from table the antioxidant potential of control sample was 32.73 ± 0.33%, whereas for treated sample it was 32.67 ± 0.29% and 32.47 ± 0.3% when treated for 30 s and 45 s respectively (Fig. 3). As evident from the obtained results that microwave has no effect on the antioxidant potential of jaggery irrespective of treatment time. Jaggery is usually produced by concentration of sugarcane juice and jaggery is the least processed product, hence it preserves almost all the bioactive components present in the sugarcane juice (Harish Nayaka et al. 2009). These, components have anti-oxidant, anti-inflammatory, anti-stress activity (Duarte-almeida et al. 2006; Ledón et al. 2003). Ma et al., (Ma et al. 2018) studied bioactivity in jaggery prepared using mucilages of plant. The authors evaluated antioxidant potential of jaggery using reducing power assay and reported that jaggery samples with and without plant mucilages showed antioxidant activities and these samples showed absorbance at 700 nm. These antioxidant activities in jaggery are clear indication of retention of phenolic compounds and flavonoids from sugarcane. Similarly, Nayaka et al., (Nayaka et al. 2009) did a comparative study on evaluation of antioxidant potential of various sugars. The authors reported that the total phenol content of jaggery was 3837 μg GAE/g which was higher among all the sugar. Further author reported that the EC50 value as revealed by DPPH scavenging essay for jaggery was 59.38 μg/ml, this value was also higher for jaggery as compared with other sugars. So, jaggery has phenolic compounds in it which are responsible for the antioxidant potential in jaggery, thus making it healthier and more nutritious among human being. But these healthier components are not disturbed by the microwave treatment used for preservation of jaggery as revealed from the data obtained in the present work.

Fig. 3.

Effect of microwave treatment on antioxidant activity of jaggery samples (T1: 30 s treatment; T2: 45 s treatment)

pH

The variation in the pH of jaggery sample after microwave treatment is shown in Table 1. The pH of untreated sample was 5.216 ± 0.22, whereas the pH of treated sample was 5.23 ± 0.24 and 5.698 ± 0.21 treated for 30 s and 45 s respectively. The microwave treatment to jaggery resulted in marginal increase in the pH value of jaggery cubes. The pH values increased with increase in microwave irradiation time from 30 to 45 s. The higher values of pH are desirable in jaggery, since low pH values of jaggery are related to formation of organic acids with the inversion of sucrose and absorption of moisture, which is not excepted in jaggery product (Chikkappaiah et al. 2017).. The treatment resulted in significant increase in pH between two treatment time and showed marginal increase in untreated sample and sample treated for 30 s. This totally depends on the exposure time of jaggery to irradiations, till 30 s the increase was marginal and after 30 s there was significant increase. Nayaka et al., (Nayaka et al. 2014) studied physical and chemical attributes of jaggery from different sugar varieties. The authors reported that the pH of jaggery was in the range of 5.66–5.87. Similar results were also reported by Guerra et al., (Guerra & Mujica 2009) for cane sugar panelas. Thus, the obtained pH in present work after microwave treatment is acceptable and it is desirable for jaggery cubes meant for human consumption. As seen from the obtained results of pH for treated and untreated samples of jaggery, microwave has a great potential to emerge as a probable preservation technique for foods like jaggery without any negative effect on the quality of jaggery and microwave does not cause inversion of sucrose in jaggery, which is usually a case of concern when jaggery is treated with direct heat / flame heat in open pans.

Microbiological analysis

The microbiological part was the important concern related to the present work, which divulge the potential of using microwave in preservation of jaggery cubes. The data observed from Table 1 shows that the values for untreated and treated samples. It is observed that the total plate count for untreated sample was higher i.e., 194 ± 48 cfu/g. However, this value was 87 ± 14 cfu/g and 13 ± 4 cfu/g for jaggery cubes irradiated for 30 s and 45 s respectively. It means, that with increase in the irradiation time, the jaggery cubes were expose to microwave environment or highest time, thus significantly reducing the microbial load in jaggery cubes. Similar effect was seen in terms to yeast and molds, the population of yeast and molds was much higher in case of untreated sample which significantly reduces after the microwave treatment. The count was 25,498 ± 2809 cfu/g for untreated sample and it significantly came down after exposure to microwave i.e., 79 ± 12 and 28 ± 7 cfu/g for samples treated for 30 s and 45 s respectively. The higher exposure time is beneficial in terms of reduction in the microbial load including yeast and mold and thus preserving jaggery for longer period of time without any ill effect on its physicochemical characteristics. Microwave irradiation results in thermal destruction of microorganism leading to their death. The amount of heat generated in the food material during microwave exposure due to direct exposure to irradiations results in increase in temperature in the food material resulting in destruction of microorganism in food material and thus reducing the microbial load in food and enhancing the shelf-life of food (Fung & Cunningham 1980; Woo et al. 2000). This effect is majorly seen when there is good correlation between the size of jaggery and the exposure time. The bigger size of jaggery needs longer exposure to microwave irradiations to achieve significant reduction in total microbial population present in jaggery, whereas higher destruction of microbes occurs in shorter exposure time, if the size of food material is smaller. As observed in the present work the size of jaggery cubes was small, hence significant reduction in total plate count (13 ± 4 cfu/g) and also in yeast and mold (28 ± 7 cfu/g) population is seen. Thus, microwave proved to be an efficient preservation method for preservation of jaggery cubes and the optimized exposure time which gave positive results in terms of reduction in microbial load, enhanced vitamin C without any negative effect on physicochemical attributes is 45 s.

Conclusion

In present study it has been investigated that the microwave treatment would be useful treatment for treating the microbial load in jaggery cubes. In our investigation, Jaggery samples were kept for microwave heating at 30 s and 45 s in domestic microwave oven having frequency 2450 MHz with power of 1 magnetron 850 W, and remarkable result has been observed. Moisture content of treated jaggery sample was 3.98 ± 0.59% and 3.86 ± 0.63% for 30 s and 45 s microwave heating respectively. Similarly, this treatment maintains the water activity at 0.6, which is considered as a safe for consumption, beyond that microbial spoilage may occur. The content of iron retains in this microwave treatment; it doesn’t affect much on the iron content present in fresh Jaggery sample. Most substantial result has been observed in case of total plate count and yeast and mold count when treated with microwave heating. In this case microwave process at 45 was found to be the best since it reduced the TPC load to 13 ± 4 from 194 ± 48 cfu/g; which is one of the outstanding observations of the present study. Also, in case on YM count, 45 s microwave treatment showed the remarkable reduction of load to 28 ± 7 cfu/g from 25,498 ± 2809 cfu/g. Each investigation was showing more reliable and suitable technique to treat the microbial load in jaggery. Hence Present investigation showed that microwave treatment for 45 s was found to be the best to treat the jaggery, which contains moisture upto 7%; since microwave works due to the dipole moment of water molecules.

Abbreviations

DPPH

2, 2-Diphenyl-2-picrylhydrazyl

ANOVA

Analysis of variance

SD

Standard deviation

Authors' contributions

PH (Conceived and designed the analysis data collection, performed the analysis), SD (Contributed data, data analysis, interpretation of data, manuscript writing), VNP (critical revision of article), AD (critical revision of article), UA (critical revision of article and final approval for the publication), YC (Critical revision, supervision, and editing).

Funding

Not applicable.

Availability of data and material

The datasets generated during the current study are available from the corresponding author on reasonable request.

Declarations

Conflicts of interest

There is No conflict of interest among the authors.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.