Juçara Pulp Drum Drying: an Alternative Technology for Value Chain Expansion

doi:10.21203/rs.3.rs-5328028/v1

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Juçara Pulp Drum Drying: an Alternative Technology for Value Chain Expansion

https://doi.org/10.21203/rs.3.rs-5328028/v1

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Juçara (Euterpe edulis Martius) is a native Brazilian Atlantic Rainforest palm tree that is commonly found in small farming communities located in well-preserved forest remnants. Although juçara is a seasonal and highly perishable fruit, requiring freezing temperatures for storage, drying presents a viable alternative to avoid the cold chain. This study evaluated the effectiveness of drum drying for juçara pulp under three conditions: (1) without carrier agents, (2) with 5% (dry basis) corn starch, and (3) with 10% (dry basis) organic rice flour. The dried juçara pulp was analyzed for its chemical composition and bioactive properties. Drum drying of juçara pulp is viable with or without the use of carrier agents., resulting in juçara flakes with a moisture content of 2.3–3.5% (wet basis). While juçara flakes maintained the same total phenolic content as the fresh pulp (p > 0.05), anthocyanin levels and antioxidant capacity were slightly reduced (p > 0.05). Despite the need for further stability analysis, the product prepared without carrier agents emerged as a promising option for producing 100% juçara pulp flakes.

Euterpe edulis Martius

Drum drying

Fruit flakes

Phenolic compounds

Anthocyanins

Antioxidant activity

- Drum drying is a viable technology for producing juçara pulp flakes, both with and without the addition of carriers.

- Total Phenolic Compounds is not significantly affected during drum drying of juçara pulp, independently of the use of carriers

- Anthocyanins and antioxidant capacity (DPPH and ABTS) is negatively affected after drum drying juçara pulp, independently of the use of carriers

The development and the strengthening of native biodiversity value chains can be used as a bioeconomy strategy through the valorization of natural resources. This is of a special importance for Juçara palm species (Euterpe edulis Martius), from the Brazilian Atlantic Rainforest, very similar to the amazonian açaí palm tree (Euterpe oleraceae Mart.). Both fruits are black-violet berries due to high anthocyanin contents, and the pulp can be consumed as the amazonian açaí, in smoothies, mixed with fruits and cereals, or applied to various food products (Barroso et al., 2019).

Conversely, most of juçara consumption comes from the unsustainable exploitation of its heart palm, which implicates in tree death. Historically, the illegal exploitation of the heart palm has placed this species on the list of threatened and endangered plants (Martinelli, 2013). Because of this, the consumption of juçara fruit is encouraged as a solution to preserve this species in the Atlantic Rainforest biome (Schulz et al., 2016).

Despite its high commercial potential, the juçara fruit pulp is still not yet well-explored on an industrial scale (Pereira et al., 2019). Yet, many small farmer communities have already implemented juçara pulp production and marketing, even on an artisanal scale, representing a potential income. These communities are located in environmentally protected areas in the state of São Paulo, along the Ribeira and Paraiba Valleys, surrounded by the most preserved Atlantic Rainforest biome remnants (Cembranelli et al., 2015).

Juçara fruit has a great nutritional potential, but since the pulping process involves the addition of water (Santana et al., 2016), its chemical properties are highly dependent on fruit processing practices. As a result, juçara pulp moisture can vary from 76 to 93% and lipid content from 2.2 to 19% (Silva et al., 2013; Bicudo et al., 2014; Santana et al., 2016 and Paim et al., 2016). Most of its nutritional interest are focused on antioxidant properties, especially anthocyanin contents, which can appear from 10.4 to 290 mg/100 g of pulp (Palm et al., 2016, Bicudo et al., 2014, Silva et al., 2013, Silva et al., 2018 e Brito et al., 2007)), with many studies dedicated to develop and understand anthocyanins-rich extracts from juçara fruit and pulp (Passos et al., 2015; Carvalho et al., 2016; Lima et al., 2019 and others). The main anthocyanin compounds founded in the juçara fruit are cyanidin 3-glu-coside and cyanidin 3-rutinoside (Brito et al 2007)

Due to the high moisture content of juçara pulp, freezing is essential for its storage, transportation, and commercialization, resulting in a major bottleneck for scaling up production in the aforementioned small farmer communities (F.P.C, personal communication).

Drying is an alternative for fruit pulp preservation with many advantages, such as higher nutrient concentrations and easier transportation, storage and commercialization. Drum drying is a method used to dehydrate materials and involves passing the material over heated drums, which evaporate the water content. It is an alternative for pasty materials, presenting several benefits, such as energy efficiency, high profitability and flexibility for different products. It is very convenient for purees, like juçara pulp, with large capacity in a continuous process (Antoniolli et al., 2022).

Stickiness occurs when drying fruit juices, due to the presence of low molecular weight compounds like sugars and organic acids, which tend to decrease glass transition temperatures and stick to surfaces under high drying temperatures (Ahmad and Nguyen, 2017). They are difficult to remove, resulting in impaired product quality, low yields, and dryer damage (Yamato et al., 2022). To mitigate stickiness, the use of high molecular weight carriers, such as starches, maltodextrins, pectins and gums, is usually required, in a concentration ranging from 3 to 20% d.b. These additives benefit the formation and detachment of the film from the cylinder, enhancing product yields (Antoniolli et al., 2022), increasing the glass transition temperatures and protecting the nutrients of the material to be dried, improving final product stability (Yamato et al., 2020; Antoniolli et al., 2022).

However, the clean label trend has driven the food industry to communicate whether a certain ingredient or carrier is not present in the final product or if the food has been produced using a more “natural” production method (Asioli et al., 2017). Clean label refers to what is seen as little processed and “natural” or “free from” negatively associated ingredients (Aschemann-Witzel et al., 2019).

The most usual process to meet the clean label demand is freeze-drying, which has been reported as a no-carriers drying method, resulting in 100% powder fruits. The main concern when applying this process comprises energy costs. Despite the development of more efficient equipment, which makes the freeze-drying drying process a viable alternative, the costs of drying some materials with high water content, like fruit pulps, are still considered too high (Asioli et al., 2017).

Açaí pulp powder is widely consumed globally, especially in the U.S., as a superfood or dietary supplement, with capsules or the actual powder used as a concentrated juice ingredient and incorporated in foods (Earling et al., 2019). Many açaí powder product variations concerning biochemical quality have been noted, including adulteration reports on the use of commercial açaí powders with very low or no açaí pulp content (Earling et al., 2019; Benatrehina et al., 2018).

Therefore, research on drying solutions for the production of fruit powders with no carriers can meet food ingredient demands and support cleaner formulations. Concerning the low sugar and acid contents of juçara pulp and its cold chain barrier, this study aims to evaluate juçara pulp powder obtained by drum drying, regarding three different carrier regimes: (1) no carrier agents, (2) 5% (dry basis, d.b.) corn starch and (3) 10% (d.b.) organic rice flour.

2.1 Juçara pulp and carrier agents

The juçara samples were harvested and depulped in the city of Sete Barras, SP (24º 22’31” S 47º56’00” W). After harvest and fruit detachment, juçara fruits were selected, sanitized and placed in plastic boxes to transport to the manufacturing place (Fig. 1). Then, berries were warmed in a water bath at 60 ºC and taken to a pulper containing 5 dm³ of water for each 8 dm³ of fruits. On average three kilograms of sanitized fruits yield one kilogram of juçara pulp. The extracted juçara pulp (JP) was then frozen at -20ºC in plastic 1 kg bags until proximal composition and bioactive compound assessments. Thereafter, 50 kg of frozen juçara pulp was collected and transported in thermal boxes to the Fruthotec/ITAL laboratory for drum drying assay. The carriers comprised regular corn starch (Ingredion, Mogi Guaçu, Brazil), and organic rice flour containing 92% starch obtained in a market in the city of Campinas, SP. Corn starch is one of the traditional carrier agents and organic rice flour was chosen as a clean label substitute.

2.2 Drum drying process of juçara pulp

The three treatments were carried out with approximately 12 kg of fresh juçara pulp each, homogenized in a colloidal mill (Meteor, REX 2-AL, São Paulo, Brazil) and fed into a rotary single cylinder dryer (Richard Simon & Sons, D139, Nottingham, England), showed in the Fig. 2. The conditions parameters were as follows: 0.15 mm clearance between the heating and applicator cylinder, pool level of 400 mL, residence time of 20 seconds, and processing temperature of 135 ºC. The process conditions were defined based on a previous study carried out with jabuticaba pulp (Nunes et al., 2020) and preliminary tests. The dried product obtained as a film was then flocculated (Fabbe, S508, São Paulo, Brazil) using a 2.5 mm sieve (8). Treatments comprised the following:

Raw material – Juçara Pulp - JP

Treatment 1 - Juçara Flakes - JF

Treatment 2 - Juçara Flakes + corn starch (5% d.b) - JFC

Treatment 3 - Juçara Flakes + organic rice flour (10% d.b) - JFR

Immediately after the drying process, the dehydrated samples were frozen at -20ºC and transported to the Horticultural Product Laboratory at ESALQ/USP (Piracicaba-SP) and stored at -20°C.

2.3 Proximal composition

Proximal composition analyses were carried out according to the AOAC (2016) for all four samples, namely the fresh juçara pulp (JP) and the three dried products (JF, JFC and JFR). Moisture content was determined by placing the samples in an air circulating oven set at 105°C until reaching constant weight. Ashes were determined by muffle incineration at 550ºC, protein contents were determined by the micro Kjeldahl method using a 6.25 factor to convert nitrogen values into protein, and soluble and insoluble dietary fibers were determined by the enzymatic-gravimetric method. Non-fiber carbohydrate contents were determined by difference. All analyses were performed for three juçara flakes (JF, JFC and JFR) and in the JP prior to drum drying.

2.4 Bioactive compounds

2.4.1 Total phenolic compounds

Total phenolic compounds were determined by the Folin-Ciocalteu method as described by Woisky and Salatino (1998). Samples (1 g) were extracted with methanol (80% v/v), filtered through filter paper and subsequently mixed with 2.5 mL of Folin-Ciocalteu’s reagent (1:10) and 2 mL of a 4% sodium carbonate solution (w/v) and maintained in the dark for 2 h. Absorbances were determined at 740 nm using a Biochrom Libra S22 spectrophotometer and the results were expressed as mg gallic acid equivalents.

2.4.2. Anthocyanins

Anthocyanin contents were estimated spectrophotometrically according to Francis (1982). A total of 1 g of samples were used for the extraction of anthocyanin compounds, mixed with 50 mL of an ethanol: 1.5 M HCl solution (85:15, v/v) and incubated for 1 h at room temperature. After this procedure, a spectrophotometer (Biochrom Libra S22) at 535 nm wavelength, which represents the absorption spectrum of the anthocyanins, was used to determine the absorbance values of the blank containing the ethanol: 1.5 M HCl solution. Total anthocyanins (TA) were calculated using Eq. 1. The results were expressed as milligrams of total anthocyanins per 100 grams of sample.

$$\:TA=\frac{ABS*F}{98.2}$$

Where TA comprises total anthocyanins; ABS is the determined absorbance and F is the dilution factor. The 98.2 factor is the acid-ethanol solvent value.

2.4.3 Antioxidant activity (ABTS and DPPH)

The antioxidant activity by the ABTS ((2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical sequestration method was carried out according to Al-Duais et al (2009). Briefly, 2 mL of an ABTS⁺ solution and 20 µL of Trolox or crude extract were added to the test tube, which was homogenized using a tube shaker in the dark for 6 minutes. Absorptions were determined on a Biochrom Libra S22 spectrophotometer at 734 nm. Each sample was analyzed in triplicate and the results were expressed as µmol Trolox equivalent (TE) per gram (d.b.).

The free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) sequestration test was measured using a method adapted by Brand-Williams et al. (1995). In brief, 500 µL of the crude extract at different concentrations were mixed with 3 mL of ethanol and 300 µL of DPPH (0.004% w/v). After 45 min of the reaction, the absorbance of the remaining DPPH was measured at 517 nm against the blanks. The antioxidant capacity of the pulp to eliminate DPPH radicals was calculated as percentage of inhibition and expressed as µmol Trolox (TE) per gram (d.b.).

2.5. Statistical analyses

The data were expressed as means ± standard deviation (M ± SD) and statistical comparisons among groups were carried out using a One-way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc test at p < 0.05.

Drum drying of juçara pulp was successfully performed both with and without carriers (corn starch and rice flour). Visually, the dried films formed were very similar across all treatments. Yields, calculated considering a dried product with 5% moisture content, were 19.25 kg/100 kg fresh pulp for no carrier tratment (JF); 20.22 and 24.52 kg/100 kg fresh pulp for treatments containing 5% (d.b.) corn starch (JFC) and 10% (d. b.) rice flour (JFR), respectively.

Table 1 presents fruit pulp composition before and after the drying process, which resulted in approximately 97% of water loss. Other components, protein, insoluble dietary fiber and soluble dietary fiber, calculated on a dry basis, were not substantially affected during the drying process. Results indicate JP with almost 13% less moisture content than that reported by Da Silva et al. (2020) which could be a result of the applied depulping methods, which incorporate warm water to the fresh fruit. Moisture seems to be significantly affected by the addition of carrier agents, although it shows a slight difference.

Table 1. Chemical composition of fresh (JP) and drum-dried juçara pulp, with and without carriers (JF, JFC and JFR).

Data are expressed by means + standard deviation (d. b. except for moisture). Means followed by the same letter in the same column do not differ statistically, according to the Tukey test (p < 0.05). Moisture, ash, lipid and protein: n = 3, insoluble dietary fiber and soluble dietary fiber: n = 2.

Carbohydrate = 100 - (lipid + protein + ashes). NS = not significantly different.

	Moisture %	Ashes %	Lipids g/100 g	Protein^ns g/100 g	Soluble Fiber g/100g	Insoluble Fiber g/100 g	Total Carbohydrates g/100 g
JP	76.6 ± 0.01 a	2.99 ± 0.02 c	29.3 ± 0.03 c	7.2 ± 0.3	3.7 ± 0.3 a	35.2 ± 0.7 b	60.51 ± 0.71 a
JFR	2.61 ± 0.20 c	3.20 ± 0.04 b	28.9 ± 0.07 d	7.3 ± 0.4	2.6 ± 0.1 b	34.4 ± 0.2 b	60.53 ± 1.09 a
JFC	2.35 ± 0.01 c	3.29 ± 0.02 b	30.9 ± 0.07 a	7.5 ± 0.2	2.5 ± 0.1 b	35.1 ± 0.1 b	58.38 ± 0.52 b
JF	3.51 ± 0.15 b	3.41 ± 0.03 a	30.4 ± 0.05 b	7.3 ± 0.3	3.4 ± 0.1 a	37.3 ± 0.1 a	58.86 ± 0.78 ab
CV%	1.17	1.63	0.40	8.52	10.37	2.2	1.34

JP (Table 1) contained higher carbohydrate contents (60.51 g/100g) and lower lipid (29.3 g/100g) and ash levels (2.99 g/100g), compared to the juçara pulp analyzed by Da Silva et al. (2020) (46.8; 39.2 and 5.1 for carbohydrates, lipids and ashes, respectively).

Most of JP consists of water, carbohydrates and lipids (Table 1). This means that for each 100 g of wet pulp, approximately 23 g corresponds to dry matter, composed basically of carbohydrates, especially fibers, and lipids. Total fiber contents can be considered high (38.9% d.b.) compared to other fruits, such as raspberries (6.5% d.b.), avocado (6.7% d.b.), banana (3.4% d.b.), and apple (2.3% d.b.) (Esteban et al., 2017). In terms of lipids, JP showed lower contents of lipids than E. olearaceae (35.0% d.b.), but higher in relation to most consumed fruits (TBCA).

Ash contents d.b. were significantly increased after the drying process (p < 0.05). Juçara flakes (JF and JFC) presented more lipids than fresh pulp, probably due to the reduction in size and pectin degradation during the drum-drying process, which facilitates oil extraction in powder form (Chia & Chong, 2015), although the differences observed were very small.

One of the disadvantages of the drying process refers to the loss of bioactive compounds due to high temperatures. Bioactive compounds and Antioxidant Activity, as well as soluble sugars, are shown in Table 2. No significant loss of total phenolic compounds (TPC) was observed for either fresh or dried juçara samples (p < 0.05) (Table 2). On the other hand, the JP (before drum dryer process) contained higher levels of anthocyanins and antioxidant activity (ABTS and DPPH).

Table 2

Bioactive compounds, antioxidant capacity and soluble sugars of fresh juçara pulp (JP), juçara flakes with rice flour (JFR), juçara flakes with corn starch (JFC) and juçara flakes with no carriers (JF)
Trat	TPC^ns	TA	DPPH	ABTS	Glucose	Frustose
JP	3840.2 ± 215.8	135.72 ± 2.1 a	1046.2 ± 1.5 a	112.9 ± 1.5 a	-	-
JFR	3646.6 ± 55.13	75.36 ± 2.5 b	206.6 ± 0.1 b	7.47 ± 0.6 b	7.11 ± 0.2 b	17.7 ± 0.3 ab
JFC	3469.1 ± 55.20	58.71 ± 2.6 c	203.8 ± 0.2 b	5.87 ± 0.6 b	8.26 ± 0.2 a	18.8 ± 0.2 a
JF	3373.4 ± 71.90	79.49 ± 2.7 b	203.8 ± 0.1 b	8.69 ± 0.4 b	6.68 ± 0.1 b	17.1 ± 0.7 b
CV%	6.71	5.69	0.36	5.12	4.35	4.76

Data are expressed as means ± standard deviations (d.b.). Means followed by the same letter in the same column do not differ statistically, according to the Tukey test (p < 0.05). JP was not assessed in terms of soluble sugars. TPC = Total Phenolic Compounds (mg/100 g); TA = Total Anthocyanins (g/100 g); ABTS = 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (mg GAE/g, GAE = gallic acid equivalent); DPPH = 2.2-diphenyl-1-picryl-hydrazyl radical (µmol TE/g, TE= trolox equivalent). NS = not significantly different.

JP is very rich in phenolic compounds (3840 mg/100g d.b.), similar to araçá (Psidium guineenses), murici (Byrsonima verbascifolia) and pitanga (Eugenia uniflora), other brazilian fruits (Stafussa et al., 2021). During the drum drying process of JP, no TPC significant losses were observed. Similar results were found by Troiani (2022) in drum drying of mango peel. However, Nunes et al. (2020) reported a small but significant loss of phenolic compounds (about 7%) in jabuticaba pulp after drum drying with corn starch. This same author also observed an increase of TPC during the drum drying of jabuticaba pulp with cassava flour as a carrier (Nunes et al., 2021).

Carrier agents used to increase glass transition temperatures are also employed to protect bioactive fruit compounds from oxidation (Santana et al., 2016). In the present study, no TPC losses were observed (Table 2), even without the addition of carrier agents (JF). This is relevant in the context of food ingredients sales, which usually use polyphenols to standardize premium botanical ingredients.

Juçara fruits are also rich in anthocyanin contents, which substantially varies during ripening: from 91 to 210 mg/100 g d.b. (Bicudo et al., 2014); as well as region of origin: from 22.81 to 660.30 mg/100 g d.b. (Borges et al., 2011). In this work, JP presented 135.72 mg/100 g d.b. of anthocyanins (Table 2), which is in line with Bicudo et al. (2014) and Borges et al (2011), but much lower than those found by Paim et al. (2016), which was 1688.10 mg/100g d.b.. This wide variation is expected, given that anthocyanin content can vary according to maturation stage, region of origin, cultivation conditions, genotype and anthocyanin extraction method (Carvalho et al., 2016; Bicudo et al., 2014 and Borges et al., 2011).

Anthocyanins contents in fruits and vegetables are inevitably affected by processing steps (de Pascual-Teresa & Sanchez-Ballesta, 2008), especially when heat is involved (Cavalcanti et al., 2011). Juçara flakes exhibited anthocyanin levels ranging from 58.71 to 79.49 mg/100g dry basis (Table 2), with a retention rate of anthocyanins between 40 and 60% compared to the fresh pulp (d.b.). It is lower than those reported in the drum drying of jaboticaba pulp: 78% of anthocyanin retention (Nunes et al., 2020); and in the spray drying of defatted juçara pulp, that can vary from 63.97 to 87.94%, according to inlet/outlet temperature and carrier agents concentration (Bicudo et al., 2014). These difference in anthocyanin retention may be a result of the protective effect of anthocyanins on lipid oxidation (Viljanen et al., 2004; Svanberg et al., 2009), since this work assessed juçara whole pulp, which are much richer in lipids than jabuticaba and defatted juçara pulp. Additionally, anthocyanin loss is significantly higher (p < 0.05) in the drying process carried out with organic rice flour compared to corn starch (Table 2). A possible explanation can be related to smaller rice starch granules ranging from 3 to 8 nm that offer higher nutrient protection against 15 nm corn starch granules (Zhou et al., 2013).

Antioxidant activity was negatively affected by the drum drying process, independent of the use carriers, this reduction in antioxidant activity was also observed during spray drying of juçara (Paim et al., 2016) and açai (Euterpe oleracea) pulp (Tonon et al., 2010). Nunes et al. (2020), also reported decreased antioxidant activity in jabuticaba pulp after the drum drying process, with the mean value of around 250 µmol TE/g d.b. Several studies have reported both decreased (Nunes et al., 2020, Chia; Chong, 2015) and increased (Chang et al., 2006) antioxidant activity after drying. Decreased antioxidant compounds contents can be a result of thermal processing, which can breakdown phytochemicals, affecting cell structure integrity and resulting in component migration and other chemical reactions (Chia & Chong, 2015). Increases, on the other hand, may be due to the release of phenolic compounds from cellular structures or the inactivation of endogenous enzymes (Chang et al., 2006). In this work, no significant differences were observed in terms of antioxidant capacity (p < 0.05) among different carriers used.

Juçara fruit is not considered rich in soluble sugars. All juçara flakes presented more fructose than glucose and no detectable levels of sucrose, which corroborates Inada et al. (2015) and Schultz et al. (2021). JF have a slight but significantly higher glucose and fructose levels than JFR and JFC, which can be a consequence of incorporating mass through the use of carriers (Nunes et al., 2020).

The increasing trend of clean label products, which exclusively utilize natural ingredients like 100% fruit powders and flakes, has challenged the traditional ingredient production within the food supply chain. This study not only provides an alternative low operational cost drying method which meets this demand, but also offers a suitable opportunity for small farmer communities to scale up their production by avoiding the cold chain.

Drum drying of juçara pulp is viable with or without the use of carrier agents. The kind of carrier agents used during the drying process did not affect antioxidant activity, protein or phenolic content compared to juçara dried pulp without carriers. Although the use of carriers revealed a slight effect in lipids, fibers, ashes and anthocyanin contents.

From the clean clean label point of view, drum drying process seems to be feasible for juçara pulp without the use of carriers. However, further studies should be conducted to assess juçara flakes stability during storage, to validate product quality.

Small farmer communities in the Ribeira Valley are already harvesting juçara fruits and exhibit the potential to scale up their production regarding the yield and high occurrence of this species in their properties. One of their main challenges is the storage capacity of juçara pulp, which demands freezing temperatures. Although drum drying technology is not compatible with their processing facilities, an outsourced facility equipped with the technology is located near farmer areas. This would lead to storage advantages and communities would be able to develop high-quality and low cost products in large batches.

Funding

The authors acknowledge the financial support from Grão Ltd, São Paulo, Brazil, under its Native Fruits Program, in a partnership with the Laboratory of Postharvest of Horticultural Products at “Luiz de Queiroz” College of Agriculture, Department of Crop Science, Food and Nutrition, University of São Paulo (Project No. 103763) and the Food Research Center, which have enabled us to carry out this study.

Acknowledgments

The authors would like to express their sincere gratitude to farmers Sidenei Carlos França, Daniel Dantas, and Gilberto Ota for generously providing juçara fruit pulp samples and for their invaluable insights into the juçara value chain and the challenges hindering its expansion. Additionally, we extend our thanks to the Institute of Food Technology (ITAL) and Allbrands Food Industry for their valuable assistance in clarifying aspects of their drum drying services.

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The authors declare no competing interests.

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Juçara Pulp Drum Drying: an Alternative Technology for Value Chain Expansion

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Abstract

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Highlights

1. Introduction

2. Material and methods

2.1 Juçara pulp and carrier agents

2.2 Drum drying process of juçara pulp

2.3 Proximal composition

2.4 Bioactive compounds

2.4.1 Total phenolic compounds

2.4.2. Anthocyanins

2.4.3 Antioxidant activity (ABTS and DPPH)

2.5. Statistical analyses

3. Results and discussion

Conclusion

Declarations

Funding

Acknowledgments

References

Additional Declarations

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