Different sources of sucrose in water kefir fermentation

Diferentes fontes de sacarose na fermentação do kefir de água

Abstract

Kefir is a functional drink with potential market growth and several beneficial properties to the human body such as intestinal microbiota balance and anti-inflammatory properties. It is produced from grains that multiply from an initial culture of microorganisms that ferment sucrose, resulting in a slightly acidic, carbonated and low-alcohol beverage. The objective of this work was to use three different substrates for the fermentation (coconut, brown, and refined sugar) and to carry out analyzes of pH, titratable acidity (TA), and soluble solids content during 48 h of the process. In the grains, microbial biomass, dry mass content, and scanning electronic microscopy (SEM) were evaluated. Five fermentations were made for each sugar. The results showed differences in the use of sugar, concluding that there was a greater difference in relation to refined sugar and the others. Drinks fermented in coconut and brown sugar showed pH and acidity values within the ranges found in the consulted literature, with grain structures that provided good adhesion of microorganisms, which was not noticed in refined sugar grains. This may be due to the white sugar’s refining process involving chemical compounds, causing the sugar to have a high sucrose content but a low concentration of minerals and nutrients that favor grain growth. However, grains did not show a high mass gain, which may have been caused by the proportion of sugar/grain (40 g/26 g), temperature (27 °C), and time (48 h) used. Considering the aforementioned parameters, this pioneering study compared coconut, brown, and refined sugars. It was concluded that coconut and brown sugars were more suitable for the fermentation of the kefir drink, due to their similarities with the desired characteristics.

Keywords:
Brown sugar; Coconut sugar; Kefir grain; Microbial biomass; Microorganism; Symbiotic drink

Highlights

Refined sugar led to the production of a drink with a high content of soluble solids

Brown sugar and coconut sugar provided grains with good adhesion exopolysaccharides (EPS)

Difference in the prevalence of microorganisms was noted with brown and coconut sugars

Resumo

O kefir é uma bebida funcional com potencial crescimento de mercado e diversas propriedades benéficas ao organismo humano, como equilíbrio da microbiota intestinal e propriedades anti-inflamatórias. Ele é produzido a partir de grãos que se multiplicam de uma cultura inicial de microrganismos que fermentam sacarose, resultando em uma bebida levemente ácida, gaseificada e com baixo teor alcóolico. O objetivo deste trabalho foi utilizar três diferentes substratos para a fermentação (açúcar de coco, mascavo e refinado) e realizar análises de pH, acidez titulável e teor de sólidos solúveis durante 48 h do processo. Nos grãos, foram avaliados biomassa microbiana, teor de massa seca e microscopia eletrônica de varredura (MEV). Foram feitas cinco fermentações para cada açúcar. Os resultados apresentaram diferenças na utilização dos açúcares, concluindo maior diferença em relação ao açúcar refinado e os demais. As bebidas fermentadas em açúcar de coco e mascavo apresentaram valores de pH e acidez dentro das faixas encontradas na literatura consultada, com estruturas de grãos que proporcionaram boa aderência dos microrganismos, o que não foi notado nos grãos de açúcar refinado. Tal fato pode ser devido ao processo de refino do açúcar branco que envolve compostos químicos, fazendo com que o açúcar possua alto teor de sacarose, porém baixa concentração de minerais e nutrientes que favorecem o crescimento dos grãos. No entanto, os grãos não apresentaram alto ganho de massa, o que pode ter sido causado pela proporção de açúcar/grão (40 g/26 g), pela temperatura (27 °C) e tempo (48 h) utilizados. Este estudo pioneiro comparou os açúcares de coco, mascavo e refinado, considerando os parâmetros mencionados. Concluiu-se que os açúcares de coco e mascavo foram mais adequados para a fermentação da bebida de kefir, por suas semelhanças com as características desejadas.

Palavras-chave:
Açúcar mascavo; Açúcar de coco; Grão de kefir; Biomassa microbiana; Microrganismo; Bebida simbiótica

1 Introduction

The search for a healthy diet is growing and among the food options that bring benefits to human health are foods considered functional. Kefir, for example, is a drink classified as functional with consumption on the rise, through research has its intake associated with modulation of the immune system, balance of the intestinal microbiota, and anti-inflammatory properties (Destro et al., 2019; Zongo et al., 2020).

Kefir is a fermented drink, slightly acidic and carbonated, with a low alcohol content resulting from the fermentation of its grains in a suitable substrate. Kefir grains are small aggregates of gelatinous mass and consist of a symbiotic association of bacteria and yeast that is wrapped in a polysaccharide matrix, kefiran. These grains multiply and ferment the medium resulting in the kefir drink (Zongo et al., 2020).

However, there is another type of kefir grain capable of fermenting sucrose in media such as fruit juice, plant extracts, and sugar water solution. Different sources of sucrose present variations in the parameters of the fermentation process, such as the duration of fermentation, growth, and multiplication of grains, in addition to producing beverages with different final characteristics. This is a drink that can be consumed right after fermentation (in natura) or be flavored with fresh fruits and herbs, being a drink option for vegan consumers, those allergic to lactose or even for those who are interested in kefir as a substitute for kefir soft drinks (Destro et al., 2019).

Given the above, the objective of this work was to evaluate different sources of sucrose and their influence on the fermentative activity of water kefir.

2 Materials and methods

2.1 Search locations

All analyzes were carried out on the premises of the Federal University of Viçosa, such as the Department of Chemistry, the Laboratory of Fermented and Distilled Beverages, the Department of Food Technology, the Laboratory of Biochemical and Fermentative Processes, the Nucleus of Microscopy and Microanalysis (NMM) and in the Packaging Laboratory.

2.2 Acquisition of kefir grains and sugars

The kefir grains were obtained by donation of artisanal production in the city of Viçosa, Minas Gerais state, available in local shops.

Sugars were purchased from local supermarkets.

2.3 Grain activation

The grains were thawed in water with an average temperature of 25 °C for 24 h, proceeding to the activation stage, where 5 g of grains were inoculated in an Erlenmeyer flask containing 500 mL of mineral water with 2 tablespoons of brown sugar (about 30 g – 6% w/v) for 7 days. The sugar solution was changed every 24 hours (Alves et al., 2021). At the end of the 7 days, it was possible to see the formation of bubbles on the surface of the solution, indicating that the grains were already active and that, from that moment on, fermentation could begin.

2.4 Fermentation

In each Erlenmeyer of 500 mL, 200 mL of water were added at an average temperature of 24 °C, and then, 30 g of sugar were added. With the homogenized solutions, the soluble solids content (SSC) of each sample was measured. The initial standard was a soluble solid content of 9 °Brix and there was no considerable variation in the amount of each type of sugar. When checking the SSC, if it was below the stipulated value, more sugar was added until reaching the desired value.

The fermentations were carried out in a batch system, each batch consisting of one Erlenmeyer for each sugar, that is, three Erlenmeyers of 500 mL containing about 30g of water kefir grains. In addition, five fermentation batches were performed, therefore, each sugar had five fermentation repetitions. A single batch lasted 48 h at 27 °C, totalizing 10 days of the process at the end of the five repetitions.

2.5 Experimental design

The experimental design was characterized as a Completely Randomized Design (CRD).

The pre-inoculum was prepared using approximately 30 g of brown sugar in a 500 mL Erlenmeyer flask, containing 200 mL of water and 30 g of water kefir grains, incubated for 48 hours in a B.O.D at 27 °C, for the fermentation process.

From the pre-inoculum, four repetitions of the fermentation process were carried out. The collection of samples was made immediately after the inoculation of the grains in the medium every 12 h from the beginning until 48 h of fermentation was completed.

The pH, SSC, and titratable acidity analyzes were performed in triplicate. The other analyzes were performed only once for each repetition.

Analysis of variance (ANOVA) and Tukey's mean comparison test, considering a 5% significance level (p < 0.05), were performed using the Minitab Statistics® software. In addition, in the Prism - GraphPad® software, linear regressions were performed regarding the analysis of soluble solids content and total acidity.

2.6 Analyzes

To monitor the fermentation process, the samples were taken every 12 hours and stored in acrylic pots with a capacity of 23 mL, kept in a freezer.

Regarding the analyzes related to the grains, for growth rate, the grains were weighed on an analytical balance - Bioscale® FA2204B-BI. For wet mass content, 5 g of grains were weighed, also in acrylic pots with a capacity of 23 mL, and stored in a refrigerator for about two days until weighing. In addition, two grains from the fermentation of each treatment were collected for the analysis of scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS).

2.6.1 Beverage analysis

The pH and SSC analyzes were carried out after collecting the samples, utilizing a pH meter – MS Tecnopon®, model RS 232, properly calibrated, readings were taken every 12 h until the end of the experiment. To assess the soluble solids content of the sample, a drop of the liquid was placed on the prism of the portable analog refractometer equipment - SBR-0032.

2.6.2 Grain analysis

To evaluate the growth rate of the grains, after each fermentation, the grains were sieved, slightly dried with a paper towel, and weighed on an electronic scale with two decimal places – Even®, BL-3200AS-BI – following the methodology of Alcides et al. (2020). Equation 1 below was applied to calculate the grain growth rate, on a wet basis, presented in percentage.

$\mathrm{\%}\mathrm{}Mass\mathrm{}gain=\mathrm{}\frac{{(m}_f-\mathrm{}{m}_i)}{m_i}\times 100$ (1)

Where:

m_i is the initial mass of the grains before fermentation;

m_f is the final mass of the grains after fermentation.

To calculate the dry mass of the grains, they were stored at 4 °C until all samples were available for analysis. Therefore, at the end of the 10 days of the process, the grains were added to porcelain crucibles and taken to a drying oven – BIOPAR®, S3SSD – remaining for 48 h at 105 °C, according to Laureys & De Vuyst (2017), except the washing step in saline solution. The empty crucibles were weighed (m3), then they were weighed with the grains and after the drying stage was completed, the crucibles with the dry grains (m2) were weighed, and in this way it was possible to obtain the dry mass content using Equation 2.

$\mathrm{\%}\mathrm{}dm=\frac{\left(m2\right)-\left(\mathrm{m}3\right)}{\mathrm{m}1}$ × 100(2)

For the SEM and energy dispersive spectroscopy (EDS) analysis, the grains were prepared according to Cesário et al., 2022. Initially, the grains were fixed in 2.5% glutaraldehyde diluted in 0.1 M phosphate buffer pH 7.2 for approximately 24 h in a refrigerator, in acrylic pots. The volume of the fixative was about 10 times the volume of the material to be fixed and this step served to keep the internal structure of the material intact, preventing more activity inside and making it possible to visualize its composition.

After the fixation step, the grains were washed 6 times for 10 min in each wash, in 0.1 M phosphate buffer pH 7.2 to maintain the osmotic balance of the grain, making it difficult to gain or lose water to the medium. Then, the grains were dehydrated in series, for 10 min, in 30, 50, and 70% ethanol, and after this last dehydration, it was possible to store the material for longer periods, with the sample immersed in the ethanol itself. To finish the dehydration step, the sequence continues with ethanol 80%, 95%, and finally, 100%, three times for 10 min each.

Then, critical point drying (CPD) was performed - CPD 030 Balzers. After drying, the grains were fixed in duly identified stubs coated with conductive double-sided tape, and covered with a thin layer of gold (15-25 nm) in the metallizer - (“Sputter Coater”) Quorum Technologies®, Q150RS. Finally, the observation was made, under vacuum, in the SEM - Leo, 1430VP, coupled to an X-ray probe (EDS) (Barão et al., 2019; Cesário, 2022; Laureys et al., 2021).

3 Results and discussion

3.1 Beverage analysis

In the first 24 h, the three fermentations showed a drop in pH value, and the most relevant drop was observed in the first 12 h for refined sugar. The observed result is related to the consumption of substrates in fermentation, producing organic acids in the medium. According to the graph below (Figure 1), in which the pH values ​​are presented in vertical bars of the application of the ANOVA, a statistically significant difference was observed between the pH values ​​at 0 h for refined sugar, as well as in 48 h, in which the value reached 3.

Figure 1
Vertical bar graph indicating pH values over the hours of fermentation and the significant differences observed. Values ​​followed by different upper and lower case letters in the bar differ from each other using the Tukey test (p≤0.05).

Water kefir is a slightly acidic drink and its pH can vary depending on factors such as fermentation time, quantity and quality of sugar, and other added ingredients (Güzel‐Seydim et al., 2023). From the pH values ​​analyzed during the 48 hours of fermentation of water kefir grains on different substrates, it was possible to observe the expected profile for fermentation.

The fermentation of water kefir in brown sugar shows a significant reduction in pH in the first 24 hours, and maintaining a pH between 4.0 and 4.5 is crucial to preserving the stability and sensory characteristics of the drink. Sucrose as a source of sugar also has a pH variation in the range of 4.00 to 4.07 (Magalhães et al., 2010; Fiorda et al., 2017; Santos et al., 2019; Güzel‐Seydim et al., 2023).

Considering, then, the pH parameter, it is noticed that in this case, the time of 12 h of fermentation approaches the ideal for the process to promote a favorable environment for the microorganisms. That is, at some point between 12 and 24 h, the pH would reach the optimal range and from that point on, the other parameters involved in the process could be evaluated. As in this study, the monitoring of fermentation was carried out only every 12 hours, if the methodology were changed to verify the parameters every six hours, the analysis could be more conclusive to the ideal time for fermentation of the grains under the specified conditions.

However, for the formation of the drink's sensory characteristics, prolonging fermentation may be appropriate as discussed in Laureys & De Vuyst (2014) who observed that the soluble solids content at the end of 48 hours is still high and even so not compromise the desirable sensorial characteristics of the drink.

As microorganisms use sugar as a carbon source for fermentation, pH values ​​tend to proportionally reduce the concentration of soluble solids in the medium. It is important to note that variations in temperature and fermentation time, in addition to the concentration of inoculated grains, can influence these results (Oliveira et al., 2018; Gamba et al., 2019; Güzel‐Seydim et al., 2023).

The values ​​obtained for total titratable acidity (TTA) for the analyzed samples are presented in Figure 2, as well as the linear regression for fermentation. The samples showed an increase in TTA values ​​during the experiment. In the coconut sugar and brown sugar samples, the increase in TTA followed a linear regression as shown in the regression coefficient in Figure 3. Refined sugar did not show a gradual increase in TTA over 36 hours, which only occurred during the 48 hours of the experiment, however, when compared to other sugars, it could be noted that this increase was relatively low.

Figure 2
Vertical bar graph showing the total acidity values ​​obtained from fermentations over the hours. Values ​​followed by different upper and lower case letters in the bar differ from each other using the Tukey test (p≤0.05).

Figure 3
Linear regression graphs expressing the obtained equations and their respective coefficients in relation to the total acidity of fermentations.

The increase in acidity of kefir in different sources of sucrose is observed in several studies, and concentrations of lactic acid may occur. It was observed that the higher acidity content at the end of the seventh day with the lower concentration of brown sugar used would be associated with the amount of acid in the medium and the consumption of the substrate by microorganisms more quickly (Magalhães et al., 2010; Conceição, 2012; Monar et al., 2014; Tavares et al., 2023).

The kefir produced, in general, has an acidic profile and this characteristic can present significant differences depending on the substrates used. In this work, a greater TTA was observed in kefir fermented in coconut and brown sugar, sugars that have a higher concentration of fructose and glucose, therefore, they provided a greater amount of substrate for the production of metabolites (essentially lactic acid) by lactic acid bacteria (LAB). What must be taken into consideration, however, are the sensory characteristics of the drink attributed to organic acids and other metabolites produced from different substrates. For refined sugar, the final acidity differed significantly from the other two sugars, allowing the assumption that there was less production of acids during its fermentation.

The initial SSC was previously established at 9 °Brix and a greater drop (Figure 4) was observed in the soluble solids content in coconut sugar, followed by brown sugar, indicating that the microorganisms in the water kefir grains were consuming considerably the provided substrate; however, there was less variation in refined sugar.

Figure 4
Vertical bar graph representing the profile of the soluble solids content (°Brix) in relation to the hours and each sugar used in fermentation. Values ​​followed by different upper and lower case letters in the bar differ from each other using the Tukey test (p ≤ 0.05).

Studies with water kefir grains in different substrates, such as soy whey and coconut sugar, inulin and xanthan gum formulations, revealed reductions in soluble solids throughout the 48-hour fermentation, indicating rapid use of the available substrate, especially in the first few 24 h. In parallel, it was observed that brown sugar reached 8.1 °Brix in up to 24 hours, similar to other studies, while refined sugar reached 5.3 °Brix, suggesting different rates of fermentation and use of substrates by microorganisms present in kefir grains of water (Oliveira et al., 2018; Alves, 2020; Pendón et al., 2022).

Although, over time, the substrate concentration was lower for grain consumption, in the case of the present study, the most considerable drop occurred in the last 24 hours of fermentation, mainly in coconut and brown sugars. This may have occurred as a result of the activity of microorganisms in the grains, which may have taken longer to stabilize in the substrate medium, taking longer to metabolize the available sugars. Furthermore, one should take into account the microbiological composition of each culture in relation to the yeasts and bacteria present to carry out the process, which may interfere with the correlation of pH values ​​with SSC found for refined sugar.

3.2 Grain analysis

The percentage of mass gain from kefir grains (Figure 5), in addition to the multiplication of microorganisms and the increase in exopolysaccharide (EPS) mass, includes the ability of the glucose polymer, dextran, to retain water in the medium (Conceição, 2012). Therefore, it is suggested that grains with higher final mass have greater water retention, which suggests that the amount of dextran produced is also greater.

Figure 5
Graph of vertical bars showing mass gain values obtained during fermentations. Values ​​followed by different upper and lower case letters in the bar differ from each other using the Tukey test (p≤0.05).

According to Graça et al. (2018), the soluble solids initially made available by the substrates are generally sugars and are used during fermentation to favor the cell growth of the grains, as well as for the production of acids. Alves (2020) found that one of his formulations with the highest cell growth was the one with the lowest acidity and, conversely, the formulation with the highest acidity was the one with the lowest cell growth.

Laureys & De Vuyst (2014) obtained an increase in grain mass, in 24 h of fermentation, from 16.4 g to approximately 28 g (about 70% increase), remaining constant until the end of the 192 hours considered. Gamba et al. (2019) observed a 3.94-fold increase in the biomass of water kefir grains for 5% (w/v) brown sugar for 24 h.

Factors related to the origin and cultivation condition may have influenced this result, such as the proportion of sugar, water, and grains, temperature, fermentation time, and excessive washing of the grains between one fermentation and another.

Notably, the percentage of dry mass varies from approximately 13% to 17% (see Figure 6). The values ​​calculated for the dry mass and water mass content demonstrated consistency with the results previously presented in the literature, corroborating the findings of several authors.

Figure 6
Graph of vertical bars showing the percentages of dry mass calculated in the grains. Values ​​followed by different upper and lower case letters in the bar differ from each other using the Tukey test (p≤0.05).

Laureys & De Vuyst (2014) found an initial increase in the dry mass of brown sugar grains, ranging from 13.8% to 16.7% after 3 hours of fermentation. Subsequently, throughout the process, this dry mass reduced to stabilize between 13% and 14% (m/m). On the other hand, Dwiloka et al. (2020) observed, during fermentation in coconut water, that the processing time significantly influenced the water rate. In a period of 48 hours, the water rate reached 97.35%, while in 12 hours a slightly lower rate was recorded, around 97.14%.

From the photos produced by the SEM (Figures 7 to 10), it was possible to observe distinctions between the structures and distribution of the fermentation grains in the sugars used.

Figure 7
Images of whole water kefir grains generated by SEM at 100x approximation. (A) whole grain resulting from fermentation in coconut sugar; (B) grain resulting from fermentation in brown sugar; (C) grain grown in refined sugar.

Figure 10
EPS observed in the photos obtained through SEM. (A) grain cultivated in coconut sugar with an approximation of 2,000x; (B) grain cultivated in brown sugar with an approximation of 3,000x; (C) grain cultivated in refined sugar with an approximation of 2,000x.

In a representative analysis, the whole grains (Figure 7) have equally irregular surfaces, and it is possible to notice that the structure of the grain cultivated in coconut sugar showed deep and apparent cracks probably resulting from the preparation process in the dehydration stage or, considering the multiplication and formation of other grains from one, this may have been a step before the separation of the grain that was discontinued during the preparation of the material for analysis. In the brown sugar grain, the presence of cracks was also noted, however, superficially in relation to the coconut sugar grain, and it can be understood that this is a grain still far from the multiplication process or less damage due to the preparation for the analysis. In both grains cultivated in coconut and brown sugar, an unbroken surface was observed, different from what could be seen in the refined sugar grain, which presented a structure with a shattered appearance at certain points, indicating the disintegration of the grain during fermentation.

In Figure 8, it is shown the images of the grains at 3,000x magnification, showing the presence of microorganisms on the external surface.

Figure 8
Images of the outer surface of water kefir grains at close to 3,000x. (A) grain grown in coconut sugar; (B) grain cultivated in brown sugar; (C) grain grown in refined sugar.

Microscopy revealed a complex association of yeasts and different forms of LAB in water kefir grains fermented in brown sugar for 24 hours. There was no prevalence of one species, as they were all uniformly distributed on the surface of the grains (Oliveira et al., 2020; Bueno et al., 2021; Laureys et al., 2021).

In the images below, one can see the presence of microorganisms such as bacteria and yeasts on the outside of the better distributed coconut and brown sugar grains, while there is a greater amount of yeast on the outside of the refined sugar grain, but arranged in a more spaced way.

In Figure 9, it is shown the microorganisms observed inside the grains. In coconut sugar, a balance was noted between the presence of yeast and different species of bacteria. In refined sugar, there is a predominance of yeast with little presence of bacteria. In brown sugar, the bacteria present are smaller than the bacteria in the coconut sugar grain, however, more notable than the bacteria in coconut sugar.

Figure 9
Microorganisms observed inside the grains, evaluated by SEM. (A) Grain cultivated in coconut sugar with 5,000x zoom; (B) grain cultivated in brown sugar with 3,000x zoom; (C) Grain cultivated in refined sugar with 3,000x zoom.

There are great variations in the distribution of microorganisms in kefir grains, with a predominance of yeast in the internal part, with groups of microorganisms, including bacteria, in the external part, and a mixture of yeast and bacteria may also occur on the external surface, with exclusive areas of each microorganism, however, defining the microorganisms present inside the cut grains becomes more difficult (Magalhães et al., 2010; Laureys et al., 2021).

In this study, the presence of yeasts was observed, LAB, and acetic acid bacteria (possibly of the genus Acetobacter). Dextran was also observed in the images captured by SEM (Figure 10) as it is characterized as the main polysaccharide that makes up and has the function of grouping microorganisms in kefir grains.

In the photos below, it is possible to identify the most predominant presence in coconut sugar and brown sugar grains, unlike the grain in refined sugar, in which it is possible to observe the formation of EPS, but in smaller quantities and irregularly. This consequence can be attributed to the fact that EPS production is related to the presence of LAB in kefir grains (Wang et al., 2022). Therefore, as a lower presence of EPS was observed in grains grown in refined sugar, this may be associated with the observation of a lower amount of LAB, responsible for the formation of EPS.

At the end of the fermentations, the presence of precipitate was observed only in the containers that contained refined sugar, and the integrity of the grains removed from the medium was compromised. Relating this information to the fact that the presence of EPS protects the structure of the grains and that in the refined sugar grain, the EPS was not identified in notable quantity, it can be concluded that the water kefir grain fermented in refined sugar had less stability and less possibility of remaining intact throughout the fermentations.

The external portion of kefir grains was composed of a structured and organized biofilm, while in the internal portion, it was possible to observe a biofilm with a lower degree of organization, but with apparent resistance capable of grouping and adhering microorganisms to the walls of the grains (Magalhães et al., 2010; Lynch et al., 2021).

4 Conclusions

The production of the kefir drink from the fermentation of the grains in sugar water can be carried out in the three substrates used in this work, with small variations in relation to the expected physicochemical characteristics. Even though the microbiological composition of the drinks was different from each other, there was no loss to the purpose of kefir consumption.

The use of refined sugar can harm the microbiota and the structure of the grains over time, confirming that the use of brown sugar and even coconut sugar are better options when evaluating the quality of the grains and the result of the drink produced, therefore, the objectives proposed for the work were achieved.

• Cite as:
  Lima, A. L. B. C., Pinheiro, L. R., Souza, B. J. F., & Pereira, A. F. (2024). Different sources of sucrose in water kefir fermentation. Brazilian Journal of Food Technology, 27, e2024012. https://doi.org/10.1590/1981-6723.01224
• Funding:
  Universidade Federal de Viçosa.

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Edited by

• Section Editor: Marta Hiromi Taniwaki.

Publication Dates

• Publication in this collection
  25 Oct 2024
• Date of issue
  2024

History

• Received
  31 Jan 2024
• Accepted
  13 Mar 2024