Sequential combination of laccase pretreatment and acid extraction for high-yield pectin production from pomelo peels

Abstract

A sequential combination of laccase pretreatment and acid extraction of pectin from pomelo peels was performed in this study. By using a laccase dose of 10 U/g, the yield and galacturonic acid content of the extracted pectin were 21.67% and 75.74%, respectively, which reflected an improvement by nearly 30% and 44% relative to the extraction process without laccase. Moreover, the degree of esterification of the extracted pectin was significantly improved as the laccase concentration was increased from 0 to 10 U/g but did not affect the molecular weight. Besides, the enzyme-treated sample displayed more than 29% loss of total lignin, in which the acid soluble lignin and the acid insoluble lignin were removed by over 15% and 33%, respectively. Scanning electron microscopy indicated that the penetration and partial destruction of the pomelo peel cell wall due to laccase might be one of the main reasons why laccase pretreatment could release pectin from cell wall complexes and increase the pectin yield in subsequent acid extraction processes.

Introduction

Pomelo (Citrus grandis (L.) Osbeck) peels (PPs) account for 30–65% of the total fruit weight depending on the stage of ripening, plant source, and genotype. PPs are generally processed into animal feed or directly disposed of as industrial wastes, which not only causes an enormous waste of natural resources but also introduces severe environmental pollution (Methacanon et al. 2014; Muller-Maatsch et al. 2016). Therefore, many studies have focused on the economic utilization of PPs to produce high-value-added compounds, such as pectins, flavonoids, dietary fibers, and carotenoids (Kongkachuichai et al. 2010). Several kinds of extraction methods, such as conventional acid extraction (Roy et al. 2018), enzyme-assisted extraction (EAE) (Wikiera et al. 2015), ultrasound-assisted extraction (UAE) (Grassino et al. 2016), and microwave-assisted extraction (MAE) (Hosseini et al. 2016; Wandee et al. 2019), have been applied to obtain pectins from various natural resources. Among them, EAE has been proven to be a favorable method due to it’s mild, efficient, and environmentally friendly properties (Wikiera et al. 2015; You et al. 2013). The principle of EAE requires specific enzymes to degrade non-target macromolecules, such as protein, cellulose, or hemicellulose, to release compounds from the cell walls and increase the yield and quality of target products. Thus, enzymes that exhibit activities toward cellulose, hemicelluloses (including xylans, mannans, and glucans) or proteins (mainly in the form of glycoproteins) have been applied to extract pectins or other components from various agricultural by-products (Domingo et al. 2015; Jeong et al. 2014; You et al. 2013).

Lignin is another major component of agricultural by-products. In our study, the lignin composition (dry weight) of PPs was approximately 24%. However, as a class of complex organic polymers, lignin has been rarely subjected to enzymatic treatment in the extraction of functional compounds from natural resources. In primary plant cell walls, lignin associates with cross-linked polymers that consist of various sugars and proteins form lignin–carbohydrate complexes (LCC) (Chai et al. 2015), which are also as known as lignocellulosic materials (Jonsson and Martin 2016). Therefore, it is hypothesized that the enzymatic degradation of lignin could disrupt the intact structure of LCC to enhance pectin extraction efficiency.

Laccase is a copper-containing polyphenol oxidase that belongs to the blue multi-copper oxidase family and has been widely found in wood-rot fungi, which is a class of microorganisms that can most effectively degrade aromatic compounds (Zheng et al. 2017). Cellulolytic enzymes, which may present different amounts of polygalacturonase, pectin lyase, and rhamnogalacturonan I lyase activities that may cause undesirable damage to the natural pectin structures (Dominiak et al. 2014; Wikiera et al. 2015). However, laccase acts only on phenols and similar molecules, but no activity on polysaccharides has been reported. Thus, based on the general action pattern of laccase, this study hypothesized that laccase pretreatment could degrade lignin and disrupt the intact structure of LCC to release other organic polymers and increase the pectin yield during the subsequent acid extraction. The main objective of this study was to investigate the effect of laccase pretreatment on the extraction efficiency and quality of pectin from PPs.

Materials and methods

Laccase

Laccase from Trametes sp. LS-10C was prepared as described in our previous work (Li et al. 2016). Crude enzyme without cellulase and pectinase activity was used in this study. The optimal temperature and pH of laccase was at 40 °C and 4.0, respectively.

Pomelo peels (PPs) preparation

The pomelos were purchased from a local market in Pinghe County, Fujian Province, China. The PPs were cut into small pieces with an average size of 4 cm² and further dried at 55 °C to reach the final moisture content of 10%. Then, the pre-dried pieces were smashed, and the grindings that filtered by 40 mesh (0.425 mm) were collected for pectin extraction. Additionally, the pre-dried pieces were further dried at 105 °C to a constant weight to calculate the dry weight.

Laccase pretreatment and acid extraction

A mixture containing 10.0 g of PPs (dry weight) and 200 mL of distilled water was prepared in a 500 mL beaker to a material-to-liquid ratio of 20:1. Then, the mixture was incubated in a boiling water bath for 10 min to deactivate potential pectin hydrolytic enzymes in the PPs. After the mixture was cooled down to 40 °C, the pH was adjusted to 4.0 with 1.0 mol/L NaOH, and laccase was added at doses of 5, 10, 20, and 40 U/g PPs. After 50 min of incubation at 40 °C, 0.5 mL of the treated sample was collected and used for lignin content determination and scanning electron microscopy (SEM) observation. The pectin in the remaining sample was extracted by following a traditional acid extraction protocol, as previously described (Methacanon et al. 2014). Specifically, the pH of the mixture was adjusted to 2.0 by 1.0 M HCl, and the acid-treated solid sample was collected and used for lignin content determination and SEM observation. During the entire incubation process, the mixture was subjected to continuous slow stirring.

Analytical methods

Laccase activity was determined according to our earlier work by using 2,2ʹ-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as the enzymatic substrate (Li et al. 2016). The pectin yield and ash content were calculated based on the direct weighing method, as described earlier (Wikiera et al. 2015). To determine the degree of esterification (DE), an acid–base titration method was used as described in the (Council 1981). The galacturonic acid (GalA) content was determined based on the colourimetric method (Liew et al. 2016) by using d-galacturonic acid as a standard. The molecular weight (MW) of the pectin samples was measured by gel permeation chromatography (Methacanon et al. 2014). Specifically, a Waters 2410 refractive index detector was used, and a set containing five different dextran standards (MW: 11,600, 23,800, 80,900, 409,800, and 667,800) was used as a standard. A method reported by the National Renewable Energy Laboratory (Katahira et al. 2013) was performed to assay the lignin content. The total lignin (TL) content, which was considered as the sum of acid insoluble lignin (AIL) and acid soluble lignin (ASL), was calculated on an extractive-free basis. For SEM observation, the samples collected from different stages were washed five times with distilled water before being dried at 105 °C. The dried samples were ground into powder and used for SEM observation following a standard sputter coating process with gold.

Statistical analysis

The data derived from triplicate experiments were expressed as means ± standard deviations (SD) and were further processed by one-way analysis of variance and post-hoc test by using IBM SPSS Statistics 20.0.0 (New York, USA). Values that do not share a letter within the figure or column are significantly different (p < 0.05).

Results and discussion

Effects of laccase pretreatment on pectin yield and chemical properties

Compared to the traditional acid extraction method, the sequential combination of laccase pretreatment and acid extraction could significantly improve the extraction efficiency of pectin from PPs. The data showed that the pectin yield obtained at the lowest dose (5 U/g) was 18.97%, which was approximately 13.8% higher than that obtained by treatment with only HCl, as shown in Fig. 1. When the enzyme usage was 10 U/g, the pectin yield (21.67%) nearly reached the maximum level (22.53%, 20 U/L), which was nearly 30% higher than that obtained without laccase pretreatment. In the case of GalA content, a similar dependence was found, as listed in Table 1. The GalA content reached 66.26% and 75.74% at laccase concentrations of 5 and 10 U/g, respectively. These values were significant improvements from those obtained by traditional acid extraction (without laccase).

Fig. 1.

The pectin yield (%, w/w) from pomelo peels (PPs) that extracted by sequential combination of laccase pretreatment and acid extraction using different doses of laccase (U/g PPs). The data are shown as means ± standard deviation (SD) (n = 3), and the bars with different letters are significantly different from each other (p < 0.05; LSD test)

Table 1.

The chemical properties of pectin that extracted from pomelo peels (PPs) by sequential combination of laccase pretreatment and acid extraction using different doses of laccase (U/g PPs)

Laccase (U/g PPs)	GalA (%)	DE (%)	Ash (%)	MW (kDa)
0	52.71 ± 2.06c	62.31 ± 1.86c	3.40 ± 0.05a	482 ± 14a
5	66.26 ± 1.66b	68.43 ± 1.42b	2.90 ± 0.05b	495 ± 18a
10	75.74 ± 1.81a	72.33 ± 1.68a	2.79 ± 0.07b	480 ± 17a
20	77.51 ± 0.92a	72.31 ± 1.11a	2.54 ± 0.07c	502 ± 15a
40	77.34 ± 1.66a	72.33 ± 0.85a	2.24 ± 0.08d	489 ± 12a

The data are shown as means ± standard deviation (SD) (n = 3), and the data with different letters are significantly different from each other (p < 0.05; LSD test)

GalA galacturonic acid, DE degree of esterification, MW molecular weights

The DE, which varies with different extraction methods and raw material sources, was related to the stabilizing property of pectin, and the DE of the extracted pomelo pectin could be significantly changed by using different pH levels and acid types (Methacanon et al. 2014). Interestingly, by using the same acid (HCl) with the equal pH value (2.0), the DE of the extracted pomelo pectin was significantly improved as the laccase concentration was increased from 0 to 10 U/g (Table 1). Moreover, laccase pretreatment could decrease the ash content but did not affect the MW of the extracted pectin.

Effects of laccase pretreatment on lignin content

In this study, both the ASL and AIL contents in the treated samples were measured, and only a small loss of lignin was found in the acid-treated samples. For example, the TL contents of the samples before (24.74%) and after acid treatment (23.97%) were not significantly different. Interestingly, %TL observed in the laccase-treated sample was significantly reduced from 24.74 to 17.51% as the laccase dose was increased from 0 to 10 U/g, as shown in Fig. 2a. At this enzyme level, more than 29% loss of TL was observed, in which the ASL and the AIL were removed by over 15% (Fig. 2b) and 33% (Fig. 2c), respectively, indicating that the action of laccase probably caused the damage of lignin. Meanwhile, when the enzyme usage was 5 U/g, at which level the pectin yield was significantly increased by almost 14% (Fig. 1), the %ASL did not change substantially. In contrast, the %AIL for the same sample was significantly reduced from 18.81% to 15.52%. This finding suggested that compared with the loss of ASL, the removal of AIL possibly exerted a more exceptional contribution to the improved pectin yield in the subsequent acid extraction process.

Fig. 2.

The total lignin (TL) (a), acid soluble lignin (ASL) (b) and acid insoluble lignin (AIL) (c) contents (%, w/w) of pomelo peels (PPs) under different treatments. Bar 1: Control (without laccase or acid treatment); Bar 2: Treatment with only acid; Bar 3: Treatment with only laccase (5 U/g PPs); Bar 4: Treatment with laccase (5 U/g PPs) and acid; Bar 5: Treatment with only laccase (10 U/g PPs); Bar 6: Treatment with laccase (10 U/g PPs) and acid. The data are shown as means ± standard deviation (SD) (n = 3), and the bars with different letters are significantly different from each other (p < 0.05; LSD test)

Effects of laccase pretreatment on the surface structure of PPs

SEM observation of the acid-treated sample showed that the rough structure of the PPs surface (Fig. 3a) was smoother after acid treatment and formed a ridge-shaped texture (Fig. 3b). Compared with the acid-treated sample, the laccase-treated sample exhibited a more irregular “concave” on the surface and formed many small lamellar granules (Fig. 3c). Further acid treatment smoothened and compressed the surface of the “concave” (Fig. 3d). The tiny pores formed on the surface of PPs may be due to the penetration and partial destruction of the cell wall caused by lignin degradation. This particular action pattern may be one of the main reasons why laccase pretreatment could release pectin from cell wall complexes and increase the pectin yield in the subsequent acid extraction process.

Fig. 3.

SEM observation of pomelo peels (PPs). Samples were collected before (a) and after (b) acid extraction without laccase pretreatment, and laccase pretreatment (c) and sequential combination of laccase pretreatment and acid extraction (d). The dried samples were ground into powder and used for SEM observation following a standard sputter coating process with gold, and pictures of the treated samples were taken at a × 1500 magnification

Conclusions

Based on the obtained data, laccase pretreatment could change the surface structure of PPs and partially degrade lignin. Thus, the pectin could be exposed to the complex cross-linked structure, resulting in a higher pectin yield. Also, the use of laccase was only a pretreatment process on the raw material rather than the primary pectin extraction process. Therefore, in theory, laccase pretreatment could be combined with other pectin extraction methods, such as UAE, EAE, and MAE, to extract pectins with high yields.

Author contributions

ZX and SS have contributed equally to the output of the research.

Compliance with ethical standards

Conflict of interest

The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.