Acid hydrolysis of Musa paradisiaca and Citrus sinensis residues for the production of glucose syrups ¹

Hidrólise ácida de resíduos de Musa paradisiaca e Citrus sinensis para a produção de xaropes de glicose

ABSTRACT

Several agro-industrial residues can be utilized for the extraction of sugars through chemical reactions. This study evaluated the extraction of total sugars and reducing sugars from banana (Musa paradisiaca) and orange (Citrus sinensis) peel residues using acid hydrolysis. It also investigated if there is a difference between the concentration of sugars present, and analyzed the effect of different sulfuric acid concentrations and reaction times. A completely randomized 2 × 4 × 2 experimental design was used to evaluate two types of waste, exposed to four concentrations of sulfuric acid (4, 6, 8 and 10% V/V) at reaction times of 2 and 4 hours. This amounted to 16 treatments with three repetitions. The experimental units consisted of borosilicate glass flasks, half submerged in a bath of vegetable oil and assembled in a rapid cooling tower from which the analyzed glucose syrups were obtained. The orange peel residues showed the highest values of reducing sugar extraction with 8% sulfuric acid concentration, presenting mean of 16.46 mg L^-1 ± SE = 0.591 and optimal hydrolysis reaction time of 2 hours. With 4 hours reaction time, the extraction of reducing sugars was higher in orange peel residues compared to banana peel residues. Both residues proved to be suitable for sugar extraction, presenting a valuable opportunity for utilization in regions where they are readily available. They can be employed in fermentation processes for bioethanol production.

Key words:
Polysaccharides; biofuel; biomass; organic matter

HIGHLIGHTS:

Effective methods were identified for extracting sugars from agro-industrial waste.

Banana and orange residues proved to be rich in exploitable sugars.

Sugar extraction offers a green alternative for biofuel production.

RESUMO:

Vários resíduos agroindustriais podem ser utilizados para a extração de açúcares por meio de reações químicas. Este estudo avalia a extração de açúcares totais e açúcares redutores de resíduos de casca de banana (Musa paradisiaca) e laranja (Citrus sinensis) usando reações de hidrólise ácida para saber se há diferença entre a concentração de açúcares presentes, analisando o efeito de diferentes concentrações de ácido sulfúrico e tempos de reação. Foi utilizado o delineamento experimental 2 × 4 × 2 completamente aleatório, avaliando dois tipos de resíduos, expostos a quatro concentrações de ácido sulfúrico (4, 6, 8 e 10% V/V) em tempos de reação de 2 e 4 horas. Totalizando 16 tratamentos com três repetições. As unidades experimentais foram compostas por frascos de vidro de borosilicato semi-submersos em um banho de óleo vegetal e montados em uma torre de resfriamento rápido, de onde foram obtidos os xaropes de glicose analisados. Os resíduos de casca de laranja apresentaram os maiores valores de extração de açúcares redutores com concentração de 8% de ácido sulfúrico, apresentando média de 16,46 mg L^-1 ± SE = 0,591 e tempo ótimo de reação de hidrólise de 2 horas. Com 4 horas de tempo de reação, a extração de açúcares redutores foi maior nos resíduos de casca de laranja em comparação com os resíduos de casca de banana. Ambos os resíduos se mostraram adequados para a extração de açúcar, apresentando uma valiosa oportunidade de utilização em regiões onde estão prontamente disponíveis. Eles podem ser empregados em processos de fermentação para a produção de bioetanol.

Palavras-chave:
polissacarídeos; biocombustível; biomassa; matéria orgânica

Introduction

Currently, the increase in food waste has become both a societal and environmental problem. The most significant environmental impacts occur during food production and consumption. In the latter stage, households play a crucial role because due to various circumstances, approximately one-third of the food ends up as waste (ONU, 2019). The basic form of solid waste management in Mexico consists of waste collection and disposal in landfills, and the disposal of waste that is likely to be reincorporated into the production system (Olay-Romero et al., 2020).

This results in a mixture of different types of waste that end up in open landfills, creating a favorable environment for the development of disease-transmitting species (Espinosa-Negrín, 2022).

According to data from the Ministry of Agriculture and Rural Development (SADER), 608,201 tons of banana were cultivated in Mexico in 2019, meanwhile 2,406,000 tons of banana were cultivated in 2021 (SADER, 2023). Regarding orange cultivation, in 2019, Tabasco ranked eighth nationwide, with a total production of 94,943 tons (SIAP, 2019). The goal of any pre-treatment technology is delignification which alters the structure of cellulose to make it more susceptible to hydrolysis (Sharma, 2020).

Due to the complexity of their bonds, acid hydrolysis has been used to treat residues with high lignin and cellulose contents (Woiciechowski et al., 2020). To facilitate the breakdown of these bonds, it is advisable to use treatments with high temperatures and extreme pH.

Using two periods of heating and agitation, Elgharbi et al. (2024) employed this technique to evaluate banana residues and cassava starch. Under three different temperature conditions, Vieira et al. (2024) evaluated the efficiency of the technique on banana and orange peels, to determine those that promote the release of reducing sugars. To test the technique on residues generated in the municipal market of Comalcalco, Tabasco, the production capacity of total and reducing sugars was evaluated in two low-value commercial residues: banana peel (Musa paradisiaca) and orange peel (Citrus sinensis). The products obtained in this study, known as glucose syrups, could serve as an energy alternative as they could be fermented to produce alcohols (Harni et al., 2021).

The present study aimed to evaluate the extraction of total and reducing sugars from banana and orange peel residues, and to analyze the effect of different sulfuric acid concentrations and reaction times. To identify the optimal conditions for maximizing sugar recovery from these residues, significant differences in extraction yields were determined.

Material and Methods

The experimental process was conducted in three stages, as shown in Figure 1. In the first stage, to facilitate their handling, necessary adjustments were made to the residues of banana and orange peels. In the second stage, acid hydrolysis was applied to depolymerize macromolecules such as cellulose, hemicellulose, and lignin. In the third stage, the extracted total and reducing sugars were quantified.

Figure 1
Methodological diagram

The collection of raw materials was carried out at the “27 de octubre “ market, located in the downtown area of Comalcalco, Tabasco. The coordinates of the site are: 18° 15ʹ 45.75ʺ N 93° 13ʹ 17.17ʺ W and 13 meters above sea level (M.A.S.L).

To carry out pretreatment, 1 kg of banana and orange peel residues was dried and distributed into 200 g packets. Moisture loss was determined gravimetrically using the constant weight technique, as suggested by the Official Mexican Standard DGN-AA-18-1975. This standard recommends a temperature of 60 °C for the drying process of solid waste. Additionally, the residues used were homogenized through a grinding operation once they were dried and crushed. Subsequently, the residues were sieved to remove coarser materials.

The extraction of sugars was through the acid hydrolysis technique, based on the method used by Delgado-Alvarado (2023), with some modifications. For this purpose, 1 g of each substrate was placed in round-bottom flasks. Subsequently, 20 mL of sulfuric acid prepared at different test concentrations were added. The samples were heated at 121 °C in a vegetable oil bath, using reflux. The reaction was controlled using a rapid cooling tower.

The quantification of total sugars was done using the reflectometry technique. For this purpose, a portable refractometer model PAL-3 was used, after being cleaned and calibrated. To determine the percentage of Brix degrees, a drop of neutralized glucosyl syrups was added to the instrument’s well, and the corresponding reading was taken.

The content of reducing sugars in the hydrolyzed syrups was evaluated using the 3,5-dinitrosalicylic acid (DNS) method, as proposed by (Miller, 1959). The DNS reagent (1 mL) was added to different test tubes, to which 1 mL of glucosyl syrup was added. This was followed by heating in a water bath for 20 min. After the reaction time, the samples were left standing at room temperature for 5 min, after which 8.0 mL of distilled water was added. Subsequently, the samples were read on a HACH UV-VIS spectrophotometer, model DR-9000, at 575 nm wavelength.

A completely randomized 2 × 4 × 2 experimental design was carried out following the methodology of Ferrer (2002), with adaptations, evaluating two types of waste exposed to four different concentrations of sulfuric acid (4, 6, 8 and 10% V/V) at reaction times of 2 and 4 h. This resulted in16 treatments with three repetitions. The experimental units consisted of borosilicate glass flasks half submerged in a bath of vegetable oil and assembled in a rapid cooling tower, from which the analyzed glucose syrups were obtained.

To analyze the quantitative results obtained in this research project, the assumptions of normality (symmetry and kurtosis) and homoscedasticity (equality of variance) were first verified. With this validation, a factorial analysis of variance (2 × 4 × 2) was conducted, considering the factors that influence the extraction of total sugars (% Bx) and reducing sugars (mg L^-1) from banana and orange residues. The factors included the following:

Type of residue: banana and orange.

Concentration of sulfuric acid: 4, 6, 8, and 10%.

Reaction time: 2 and 4 hours.

For the analysis of data obtained from the 2 × 4× 2 factorial experimental design, a three-way analysis of variance was conducted at p ≤ 0.05. The interaction among variables was analyzed to determine if the combination of factors had a significant effect, and interaction plots were used to visualize the results. The coefficient of variation was calculated to assess the relative variability of the data in relation to the mean.

The obtained data were evaluated using the Statgraphics Centurion XVII software (Statgraphics Technologies, Inc., 2015). All models are based on the following general model (Eq. 1):

$$Y_{ij}=\mu +{\alpha }_i+{\beta }_j+{\left(\alpha \beta \right)}_{ij}+{\epsilon }_{ijk}$$ (1)

where:

Y_ij​ - value of the dependent variable for the i-th time, j-th type of residue, and k-th experiment;

μ - overall mean of the dataset;

α_i​ - effect of the reaction time;

β_j​ - effect of the H₂SO₄ concentration;

(αβ)_{ij​ -} interaction between the factors α and β; and,

ε_ijk - random error associated with the i-th time, j-th type of residue, and k-th experiment.

Results and Discussion

During the quantification of reducing sugars, a coefficient of determination R² of 0.982 was obtained, indicating a strong relationship between the concentration of reducing sugars and the absorbance measured (Figure 2). In this context, Burgos-Montañez (2020) emphasized that a coefficient of determination close to 1 indicates a good correlation between the data when relating concentration with absorbance, as well as high reliability in the obtained results. This supports the effectiveness of the experimental procedure used to prepare the analysis solutions.

Figure 2
Calibration curve of reducing sugars in function of absorbance

In this study, the equation utilized to calculate the concentrations of reducing sugars was derived from the fitted model specifically designed for this purpose (Eq. 2).

$$RS=60.5872+2,488.95\times A$$ (2)

where:

RS - reducing sugar (mg L^-1); and,

A - absorbance (nm).

The analysis of variance (Table 1) showed highly significant differences in the interaction between sulfuric acid (H₂SO₄) concentrations, reaction times (2 and 4 hours), and type of residues (banana and orange) in relation to the percentage of total sugar extraction. With p ≤ 0.001, an increase in the average extraction of total sugars was observed as the concentration of H₂SO₄ (4, 6, 8, and 10%) (factor C) and reaction times (2 and 4 hours) (factor B) increased. It was also found that the average extraction did not vary significantly for H₂SO₄ concentrations of 4% (2 hours = 8.38% BX ± SE = 0.48, 4 hours = 8.53% BX ± SE = 0.4871) and 8% (2 hours = 12.8% BX ± SE = 0.48, 4 hours = 13.3% BX ± SE = 0.48) at both reaction times. The highest extraction of total sugars was observed using a 10% concentration of H₂SO₄ and a reaction time of 4 hours, with an average value of 17.5% BX ± 0.48, followed by a reaction time of 2 hours with 13.97% BX ± SE = 0.48 (Figure 3).

Table 1
Analysis of variance for concentration of total sugars

Source	Sum of squares	Degrees of freedom	Mean square	F-ratio	p-value	Coefficient of variation
Total sugars Main effects						26.19%
A: Type of residue	7.98063	1	7.98063	4.20	0.0458
B: Reaction time	27.8256	1	27.8256	14.66	0.0004
C: Concentration H2SO4	418.937	3	139.646	73.56	0.0001
Interactions
AB	40.6406	1	40.6406	21.41	0.0001
AC	4.53688	3	1.51229	0.80	0.5018
BC	20.6969	3	6.89896	3.63	0.0192
ABC	23.6119	3	7.87063	4.15	0.0108
Residuals	91.12	48	1.89833
Total	635.349	63

Figure 3
Total sugars in function of sulfuric acid concentration at two reaction times

According to Broda et al. (2022), in hydrolytic processes that employ high concentrations of acid, the hemicellulose fraction of the residues may be depolymerized due to the action of the acid, thereby maximizing sugar recovery. This explains why maximum sugar extraction is obtained by increasing the concentration of H₂SO₄ to 10% at reaction times of 2 and 4 hours.

In terms of average total sugar extraction, the analysis of variance indicated highly significant differences (p ≤ 0.001) between reaction time (B) and type of residue (A). The results showed that banana residues recorded the highest average extraction of total sugars with a hydrolysis time of 4 hours (13.93% BX ± SE = 0.34% BX), surpassing orange residues (11.63% BX ± SE = 0.34). Additionally, the second highest average of total sugars was obtained from orange residues with a hydrolysis time of 2 hours (11.90% BX ± SE = 0.34), exceeding those obtained from banana residues (11.01% BX ± SE = 0.34). Furthermore, it was demonstrated that there is a greater average extraction of total sugars at a hydrolysis time of 2 hours (11.90% BX) compared to 4 hours (11.63% BX) in orange residues (Figure 4).

Figure 4
Interaction of the factors residue type (banana and orange) and hydrolysis time (2 and 4 h) on the average extraction of total sugars

Regarding the interaction between the type of residue (A) and sulfuric acid concentration (C), no statistically significant differences were found (p = 0.5018).

The analysis of variance (Table 2) indicated highly significant differences among the interactions of H₂SO₄, reaction times, and type of residues regarding the percentage of reducing sugar extraction. The analysis of variance showed highly significant statistical differences (p ≤ 0.001) in the extraction of reducing sugars when factors such as residue type (A) and reaction times (B) interacted. It was observed that orange residues exhibited the highest average extraction of reducing sugars, both at hydrolysis times of 2 and 4 hours, with values of 13.66 mg L⁻¹ ± SE = 0.418 and 10.81 mg L⁻¹ ± SE = 0.418, respectively. In second place, banana residues at 4 hours showed a value of 10.20 mg L⁻¹ ± SE = 0.418. Conversely, the lowest average extraction of reducing sugars was obtained from banana residues at a hydrolysis time of 2 hours, with a value of 2.39 mg L⁻¹ ± SE = 0.418 (Figure 5). These results demonstrate the possibility of utilizing banana residues in regions where banana production exceeds that of oranges. For example, in the states of Tabasco and Chiapas, which are considered the main producers in the country, approximately 480,000 tons of lignocellulosic waste are generated annually (Aguilar et al., 2019). These findings underscore the importance of considering the availability of residues and their potential value in the industry, especially in regions with significant banana production.

Table 2
Analysis of variance for concentration of total sugars

Source	Sum of square	Degress of freedom	Mean squar	F-ratio	p-value	Coefficient of variation
Reducing sugars Main effects						63.20%
A: Type of Residue	621.941	1	621.941	131.36	0.0001
B: Reaction time	76.8471	1	76.8471	16.23	0.0002
C: Concentration H2SO4	147.044	3	49.0145	10.35	0.0001
Interactions
AB	399.55	1	399.55	84.39	0.0001
AC	518.237	3	172.746	36.49	0.0001
BC	7.0961	3	2.36537	0.50	0.6843
ABC	86.1227	3	28.7076	6.06	0.0014
Residuals	227.259	48	4.73457
Total	2084.1	63

Figure 5
Interaction between residue type (banana and orange) and hydrolysis time (2 and 4 h) on the average extraction of reducing sugars

Regarding reaction time (A) and sulfuric acid concentration (C), no significant differences were found (p = 0.6843) (Table 2). However, greater extraction was recorded at hydrolysis times of 4 and 2 hours as the concentration of H₂SO₄ increased from 4 to 8%. At both hydrolysis times, a significant decrease was also observed when the concentration of H₂SO₄ increased to 10%. At 8% concentration of H₂SO₄ and hydrolysis time of 4 hours, the highest average value of extracted reducing sugars was observed, with a value of 13.55 mg L⁻¹ ± SE = 0.591. As for the interaction between the remaining factors (sulfuric acid concentration and hydrolysis times of 2 and 4 hours) in the extraction of reducing sugars, the lowest values were obtained (Figure 6).

Figure 6
Interaction between reaction time and sulfuric acid concentration in the extraction of reducing sugars

This result is consistent with the findings of Islas et al. (2012), who identified an acid concentration limit for increasing the percentage of hydrolysis when working with Beta vulgaris L. It was found that at higher percentages of hydrolysis, the sugar concentration decreased, which led to the conclusion that sulfuric acid at concentrations greater than 0.5 N does not significantly affect the sugar release process, possibly because larger areas within the substrate matrix are inaccessible or because the size of the sugars is too large to be incorporated into the reaction medium.

Furthermore, Herrera et al. (2003) reported that the acid hydrolysis of sorghum yields sugars such as xylose and glucose, as well as minor components like acetic acid and furfural. However, the concentration of these components, especially xylose, reached a maximum production at acid concentrations of 6% and a time of 60 min. After this time, production decreased drastically, suggesting that during the diffusion of reaction products, some large oligomers could not penetrate the substrate matrix and remained trapped within it, thereby hindering their incorporation into the reaction medium.

In the extraction of reducing sugars, the analysis of variance (Table 2) showed highly significant differences (p ≤ 0.001) in the interaction between residue type (A) and H₂SO₄ concentration (C). The results demonstrated greater extraction of reducing sugars as the concentrations of H₂SO₄ increased in both residues, except for orange residues, where the extraction of reducing sugars significantly decreased at a concentration of 10% H₂SO₄. The highest average value of reducing sugars (16.46 mg L⁻¹ ± SE = 0.591) was obtained from orange residues with an 8% concentration of H₂SO₄, followed by 6 and 4% concentrations of H₂SO₄, with average values of 13.63 mg L⁻¹ ± SE = 0.591 and 13.08 mg L⁻¹ ± SE = 0.591, respectively. Considering banana residues, the highest extraction of reducing sugars (average value of 9.38 mg L⁻¹ ± SE = 0.591) was observed using a 10% concentration of H₂SO₄, followed by 8, 6, and 4% concentrations of H₂SO₄, with average values of 7.9 mg L⁻¹ ± SE = 0.591, 5.1 mg L⁻¹ ± SE = 0.591, and 3.40 mg L⁻¹ ± SE = 0.591, respectively (Figure 7). In this regard, Orozco (2017) stated that increasing the reaction time and acid concentration during hydrolysis can result in an increase in the sugars present in the hydrolyzed material. However, when hydrolyzing fresh orange fruits with acid volumes exceeding 0.75% v/v, no increase in reducing sugars was observed, possibly due to the degradation of sugars into other products such as furfural. Nonetheless, depending on the biomass used, it may be necessary to employ a higher acid concentration if the substrate has a high lignin content.

Figure 7
Interaction between residue type and sulfuric acid concentration in the extraction of reducing sugars

Conclusions

1. The orange peel residues showed the highest values of reducing sugar extraction with 8% sulfuric acid concentration, presenting average of 16.46 mg L^-1 ± SE = 0.591 and an optimal hydrolysis reaction time of 2 hours.

2. With 4 hours of reaction time, the extraction of reducing sugars was higher in orange peel residues compared to banana peel residues.

3. Regarding the extraction of total sugars, the highest values were found in banana residues, with average of 15.88% B_X using 10% sulfuric acid concentration and optimal hydrolysis reaction time of 4 hours.

Acknowledgements

The authors of this article thank the Tecnológico Nacional de México campus Comalcalco and the M.I.P Abimael Aguilar Oliva for the use of the institution’s laboratory.

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• ¹ Research developed at Comalcalco, Tabasco, México

Supplementary documents

• There are no supplementary sources.

Funding statement

• The authors declare that no funding was received from any institution.

Edited by

• Editors: Lauriane Almeida dos Anjos Soares & Walter Esfrain Pereira

Data availability

There are no supplementary sources.

Publication Dates

• Publication in this collection
  03 Feb 2025
• Date of issue
  June 2025

History

• Received
  11 Jan 2024
• Accepted
  04 Dec 2024
• Published
  16 Dec 2024