Enhancement of cell disruption and selective recovery of intracellular components from selenium-enriched Konjac fly powder assisted by pulsed electric fields

Abstract

This study investigated the effect of pulsed electric field (PEF, E = 0–6 kV/cm, t = 0–5.65 ms) on cell disruption and selective extraction of intracellular components from selenium-enriched Konjac fly powder, in comparison with high pressure homogenization (HPH). The intracellular components extractability and cell disruption degree were evaluated by extraction indexes and cell disruption index, respectively. Results showed that PEF significantly improved cell disruption and components release. The extraction indexes increased with higher E and t. The extractability order was ionic components > carbohydrates > proteins, while the maximum selenium level was obtained at 1.13 ms. Compared to HPH, PEF favored carbohydrates extraction over proteins. For example, at 3 kJ/g, PEF gave a selectivity index (S) ≈ 3.0 (4 kV/cm) and ≈ 4.2 (6 kV/cm), while HPH gave S ≈ 2.3. Non-linear relationships between extraction indexes and cell disruption index reflected PEF differential effects on cell membranes and walls.

Highlights

• •PEF significantly improve cell disruption degree and intracellular components extractability.
• •Correlation between extractability and cell disruption degree was discussed.
• •The extractability was evaluated by extraction indexes of intracellular components.
• •PEF allow selective extraction of more carbohydrates than proteins.

1. Introduction

Konjac (Amorphophallus Konjac K. Koch), a perennial herb of the Araceae family, has been recognized by the United Nations Health Organization as one of the top ten health foods (Luo et al., 2022). It has attracted considerable attention in recent decades due to its high nutritional content, including a variety of vitamins and potassium, phosphorus and other mineral elements (Reang et al., 2023). Konjac glucomannan in extracts has also been shown to regulate the balance of gut microbiota in the human body (Kapoor et al., 2024). These properties have positioned konjac as a promising candidate for the production of novel foods, feeds and desiccants characterized by low calorie, low fat, and high cellulose content (Deng et al., 2024).

Currently, the main processed product is konjac mannan, which accounts for over 60 % of the total global export volume (Srzednicki & Borompichaichartkul, 2020). Konjac fly powder (KFP), a by-product consisting of light and small particles generated during the processing of konjac mannan, accounts for 30 %∼40 % of the mass fraction of konjac mannan (Nie & Gao, 2022). It has been reported that China's annual production of konjac mannan is approximately 25,000 tons (Srzednicki & Borompichaichartkul, 2020), resulting in a significant amount of by-products due to the high consumption of konjac. KFP is rich in starch polysaccharides and proteins, with a crude protein content of up to 20 % and a total of 17 amino acids, and further degradation can produce a variety of bioactive peptides, making it a valuable source of natural plant proteins (Nie & Gao, 2022). In addition to the basic nutrients of regular konjac, selenium-enriched KFP (≥ 0.15 mg/kg Se), which is rich in the essential trace element selenium, offers potential health benefits to the human body, including antioxidant, cancer prevention, treatment of Keshan disease and Kashin-Beck disease, and human resistance enhancement (Bai et al., 2025; Han et al., 2024; Wen et al., 2022). However, KFP often used as agricultural waste in the industrial food processing and production, resulting in wasted resources and environmental pollution (Srzednicki & Borompichaichartkul, 2020). Therefore, the extraction of the active compounds of KFP by a suitable method favors an increase in the efficiency of resource utilization.

Due to the multilayered cell structure of KFP, these active compounds located in different parts of the cell are protected by the rigid cell wall and membranes surrounding the cytoplasm and internal organelles, making them less readily available (Zhang et al., 2020). The most commonly used extraction methods for obtaining intracellular compounds from KFP include alkali extraction and acid isolation (Mao, Lv, & Zhang, 2014), enzyme lysis (Fu, Ma, Huang, & Lan, 2024) and hot water extraction (Yang et al., 2018). Although these methods can obtain most of intracellular compounds, they are gradually being replaced by novel extraction technology due to the disadvantage of long extraction time, high temperature, easy degeneration and leading to the loss of its activity. Physical treatments such as ultrasound (Delran et al., 2023; Zhang et al., 2019) and microwave (Pongmalai et al., 2015), which are known to be environmentally friendly and safe, have received considerable attention in recent decades. Compared to traditional methods, physical treatments not only offer green and efficient processes, but also save time and energy. They can also significantly improve the selective recovery of intracellular compounds, enabling a faster and more efficient release at low temperature to limit the degradation of extracts and reduce the impact on their functional properties and nutritional value (Postma et al., 2016).

Pulsed electric fields (PEF) are considered as a mild physical pretreatment assisted extraction technology (Carullo et al., 2018; Zhang et al., 2020). During PEF processing, the biomass is exposed to high intensity electric fields in short pulses, causing pore formation in the cell membrane and allowing the exchange of molecules (Zhou et al., 2022). The field strength, pulse frequency, and duration affect cell structure (Gateau et al., 2021), but membrane resealing can occur within 30 s (Bodénès et al., 2019). Properly adjusted PEF treatment can reversibly increase membrane permeability, facilitating compound exchange without killing the cells (Gateau et al., 2021). Previous studies have reported that PEF treatment can increase the extraction efficiency of intracellular compounds (e.g. carbohydrates and small molecular weight of water-soluble proteins) from microalgal biomass (Carullo et al., 2018; Postma et al., 2016), but the extraction of higher molecular weight components or those more tightly bound to the intracellular structure requires a more intensive cell disruption technique, such as high pressure homogenization (HPH) treatment (Katsimichas et al., 2023). However, to the best of our knowledge, the correlations between PEF-induced damage to KFP cells and the selective extraction of intracellular compounds have not been discussed in detail.

The aim of this study was to investigate the effect of PEF treatment on enhancing cell disruption and selective extraction of intracellular components from selenium-enriched KFP. The correlation between the extractability of intracellular compounds (ionic components, proteins, carbohydrates, and total selenium) and the degree of cell disruption caused by PEF was evaluated for the first time. The cell disruption degree was estimated by microstructure analysis using scanning electron microscopy (SEM) and measuring cell disruption index. The extractability of intracellular compounds during PEF treatment was monitored by changes in the extraction indexes of ionic components, protein, carbohydrate, and total selenium. Moreover, this study also discusses the extraction selectivity of PEF for carbohydrates and proteins. The obtained data were compared with those from HPH treatment.

2. Materials and methods

2.1. Chemicals and materials

Konjac fly powder (KFP), a by-product of the dry production of konjac mannan, was purchased from the Jiangjiaguan Village Konjac Farmer Cooperative (Shanxi, China), and stored at −20 °C until use. The moisture content of KFP was approximate 8.1 wt%. The primary components of KFP include 224.20 ± 0.04 mg/g dry matter (DM) of proteins, 405.00 ± 1.94 mg/g DM of carbohydrates and 0.29 ± 0.02 mg/kg DM of total selenium, along with a variety of other components, such as alkaloids, polyphenols and minerals, present in smaller quantities.

d-glucose (98 %) standard was provided by Aladdin (Aladdin Industrial Corporation, Shanghai, China) and bovine serum albumin (BSA Albumin Fraction V, 98 %) standard was provided by Feiyangbio (Biofroxx, Germany). Bradford Dye Reagent was purchased from Ourchem (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). All other chemicals were of analytical grade (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China).

2.2. Experimental design

In the present study, the cell disruption of KFP through PEF treatment at various electric field strengths was compared with HPH treatment. The untreated (0 kV/cm) suspension was also analyzed as the control experiment. The procedural steps are illustrated in Fig. 1a.

Fig. 1.

Schematic presentation of extraction procedures (a), schematic diagram of pulsed electric fields (PEF) (b) and high pressure homogenization (HPH) (c) devices.

2.2.1. Preparation of the samples

The suspension with the concentration of 5 % DM was always used in this study. Briefly, 20 g of KFP was dissolved in 400 mL of deionized water at 25 °C with magnetic stirring at 200 rpm. After blending, the conductivity of sample was measured. In this study, the initial conductivity value was approximately 1500 μS/cm.

2.2.2. Pulsed electrical fields (PEF) treatment

A PEF generator (THU-PEF4, Wuhan Xinputian laboratory equipment co., ltd., China) was utilized, featuring a continuous flow treatment chamber comprising two parallel stainless-steel electrodes spaced 3 mm apart (Fig. 1b). The suspension was conveyed through a silicone tube with an inner diameter of 5 mm and introduced into the cylindrical treatment chamber with 3 mm between the two metal electrode plates and a chamber volume of 0.02 mL, using a peristaltic pump with a flow rate of 1.43 mL/s. Prior to operation, the bubbles in the system should be removed by pumping in a portion of the sample. The voltage of PEF system was monitored by a digital storage oscilloscope (TEKWAYDST1102B, Hangzhou, China). The PEF treatment protocols involved the application of a frequency of 1.01 kHz and a fixed bipolar square wave with a pulse width of 40 μs. The electric field strengths, E, were changed from 2 to 6 kV/cm. The single cycle time was 280 s, and the total processing time was approximately 2800 s for 10 cycles, with an effective electrical treatment time of 5.65 ms. The inlet and outlet temperatures of the sample were monitored, and an ice water bath was used to keep the temperature after each cycle below 40 °C.

The suspension after each pulse were collected for further characterization analysis. The specific energy consumption, W (J/g DM) was calculated using the following Eqs. (1), (2) (Gao et al., 2022; Wu et al., 2020):

$$t=\left(V\times f\times W_p\times N\right)/S$$ (1)

$$W=E^2\times t\times \sigma /m$$ (2)

where t represents the effective PEF treatment time (ms), V represents the volume of the chamber (mL), f represents the pulse repetition rates (s⁻¹), W_p represents the pulse width (s), N represents the sample cycles, S represents the flow rate (mL/s), E represents the electric field strength (V/m), σ represents the conductivity of the suspensions (S/m), and m is the biomass concentration, g/mL.

2.2.3. High pressure homogenization (HPH) treatment

The HPH treatment was conducted using a ^LtFB high pressure homogenizer (Litu Ultra High Voltage Equipment Co., Ltd., Shanghai, China) (Fig. 1c). The homogenizer has an average throughput of 5 L/h. The homogenizing pressure, P, was set on 100 MPa. The number of passes, N, ranged from 0 to 10, respectively. The initial temperature of suspensions before HPH treatment was maintained at ambient temperature, and the final temperature during HPH treatment never exceeded 35 °C. Prior to each treatment, the suspension was re-cooled to room temperature using an ice-water bath to prevent the temperature elevation, which could otherwise cause protein denaturation.

Specific energy consumption, W (J/g DM), for HPH treatment was calculated using the following Eq. (3) as described by (Zhang et al., 2021):

$$W=P\times N/C_m\times \rho$$ (3)

where C_m is the suspension concentration, and ρ is the density of the suspension (g/m³).

2.3. Analysis of suspension

2.3.1. Ionic components

The ionic components of the suspension were measured using a conductivity meter (Mettler-Toledo SevenCompact™ conductivity meters S230, Shanghai, China) at 25 °C.

2.3.2. Particle size distribution (PSD)

The PSD can reflect the changes of cell size before and after treatments, and that was monitored in the range of 0.01–3000 μm by laser diffraction with the Malvern Mastersizer 3000 (MS3000, Malvern, UK). A refractive index of 1.45 and an absorption index of 0.100 were set for the measurements. Before determination, the suspensions were thoroughly mixed and added until the shading fell within the range of 10 %∼20 %. The PSD of cells was calculated using the original Malvern software.

2.3.3. Scanning electron microscopy (SEM)

A ZEISS Gemini SEM 300 (ZEISS, Germany) scanning electron microscopy was utilized to analyze the microstructure of KFP cells. In brief, the suspension obtained after PEF or HPH treatment was affixed to the conductive adhesive and then gold-sputtered for 45 s at 10 mA using a Quorum SC7620 sputter coater. SEM images were captured at various magnifications (500×, 1000×, 5000×, 20,000×, respectively) with an accelerating voltage of 3 kV.

In this line, the fractal dimension (FD) of cells before and after different treatments was calculated to quantify the cell disruption degree (Langrish et al., 2006; Pongmalai et al., 2015). At least 5–8 SEM images of each sample at 5000× magnification was binarized by Image J software (Version 1.53e, National Institutes Health, Bethesda, MD, USA), and calculated by Eq. (4).

$$\mathit{FD}=\underset{\epsilon \to 0}{\lim }\left[\log N\left(\epsilon \right)/\log \left(1/\epsilon \right)\right]$$ (4)

where FD denotes fractal dimension; N denotes the number of grids that cover the fractal; ε denotes the length of a grid side.

2.4. Analysis of supernatant

The cell suspensions obtained after different treatments were centrifuged using a Centrifuge 5424R (Eppendorf, Germany) at 8000 rpm for 5 min. The supernatants were collected for further chemical analyses. All analyses were based on color reaction and absorption spectra that were measured using a UV/Vis spectrophotometer AOELAB A580 (AOELAB, Shanghai, China).

2.4.1. Protein content

The water-soluble protein content, C_p, was determined using Bradford method (Bradford, 1976). In brief, 1 mL of diluted supernatant was mixed with 5 mL of Bradford Dye Reagent. After vertex for 10 s, the mixture was allowed to stand for 5 min at 25 °C. Subsequently, the absorbance was measured at 595 nm, and the value of C_p was calculated using a calibration curve constructed with BSA range from 0 to 250 μg/mL as the standard.

The total protein content of KFP raw material was measured using a Kjeldahl nitrogen method and the conversion factor N = 6.25 was selected to convert the nitrogen level to protein. The purity of protein in powder was calculated according to previous assays (Wu et al., 2020).

2.4.2. Carbohydrate content

The water-soluble carbohydrate content, C_c, was determined using the phenol‑sulfuric acid method with some modifications (Zhang et al., 2021). Briefly, 1 mL of appropriate diluted supernatants were mixed with 1 mL of phenol solution (5 %, w/v) and 5 mL of 98 % concentrated sulfuric acid. The mixture was thoroughly mixed using a vortex and kept for 30 min. The absorbance was measured at 490 nm and the value of C_c was calculated using a standard curve prepared with d-glucose range from 0 to 100 mg/L as a standard.

2.4.3. Selenium content

Total selenium content, C_Se^t, was determined using a hydride generation-atomic fluorescence spectrometer (Beijing Haiguang Instrument, China) (Yin et al., 2024). In brief, the supernatant was first freeze dried at −60 °C for 72 h under 0.5 MPa. Then, 0.2 g of dry sample were introduced into a digestion tube and mixed with 8 mL of HNO₃, subsequently digested in a multiwave PRO oven (Anton Paar, Austria). After digestion, the sample underwent heating at 180 °C until the final volume was reduced to 1 mL. The digested mixture was diluted to 10 mL using HNO₃ (2 %, v/v) for further analysis. Furthermore, the inorganic selenium content, C_Seⁱ, was measured using the procedure described below. Two hundred milligram sample was mixed with 5 mL of ultrapure water, followed by 20 min of ultrasound treatment. The mixture was heated in a water bath at 70 °C for 10 min and subsequently centrifuged at 8000 rpm for 5 min. The supernatant was extracted with cyclohexane (1:1, v/v), and 2 mL of the aqueous phase was introduced to a digestion tube, and subsequent procedures were consistent with the determination of total selenium content as previous described. The organic selenium content, C_Se^o, was calculated as following Eq. (5) described:

$$C_{\mathit{Se}}^{\mathrm{o}}=C_{\mathit{Se}}^t-C_{\mathit{Se}}^i$$ (5)

where the superscripts o, t and i correspond to the organic, total and inorganic selenium content (mg/kg), respectively.

2.5. Extraction indexes

Based on the measured values of σ, C_p, C_c, C_Se^t, and FD, the extraction indexes for ionic components, Z_i, protein, Z_p, carbohydrate, Z_c, and total selenium, Z_Se, and cell disruption index, Z_d, were defined as the following Eqs. (6), (7), (8), (9), (10):

$$Z_i=\left(\sigma -{\sigma }_{\min }\right)/\left({\sigma }_{\max }-{\sigma }_{\min }\right)$$ (6)

$$Z_p=\left(C_p-C_p^{\min }\right)/\left(C_p^{\max }-C_p^{\min }\right)$$ (7)

$$Z_c=\left(C_c-C_c^{\min }\right)/\left(C_c^{\max }-C_c^{\min }\right)$$ (8)

$$Z_{\mathit{Se}}=\left(C_{\mathit{Se}}^t-{C_{\mathit{Se}}^t}^{\min }\right)/\left({C_{\mathit{Se}}^t}^{\max }-{C_{\mathit{Se}}^t}^{\min }\right)$$ (9)

$$Z_d=\left({\mathit{FD}}^{\max }-\mathit{FD}\right)/\left({\mathit{FD}}^{\max }-{\mathit{FD}}^{\min }\right)$$ (10)

where the superscripts min and max refer to the minimum and maximum values, respectively. Here, the values of C_p^max (224.20 ± 0.04 mg/g DM) and C_c^max (405.00 ± 1.94 mg/g DM) obtained from raw material, while the values of σ ^max (2120.33 ± 2.05 μS/cm), C_Se^max (0.27 ± 0.03 mg/kg DM) and FD^min (1.65 ± 0.07) obtained from HPH treatment at 100 MPa after 10 passes.

2.6. Statistical analysis

All experiments and measurements were repeated at least three times. Data are expressed as mean ± standard deviation using SPSS 26 software. The error bars shown on the figures correspond to the standard deviations. Data analysis was conducted using single-factor ANOVA to determine the differences among groups. The statistical significance was defined as p < 0.05.

3. Results and discussion

3.1. Effect of PEF treatment on extractability of intracellular components

The extractability of PEF treatment at different electric field strengths (E = 0–6 kV/cm, t = 0–5.65 ms) was evaluated by monitoring the extraction indexes of ionic component, Z_i, protein, Z_p, carbohydrate, Z_c, and total selenium, Z_Se, in comparison to HPH treatment (p = 100 MPa, N = 10). As shown in Fig. 2, the lowest extraction indexes of intracellular components (Z_i ≤ 0.292, Z_p ≤ 0.005, and Z_c ≤ 0.055) were observed in untreated samples (E = 0 kV/cm) due to spontaneous cell lysis. The application of PEF and HPH treatment significantly increased their extractability compared to untreated samples (p < 0.05) (Fig. 2). For example, extraction for E = 6 kV/cm at t = 5.65 ms resulted in Z_i = 0.887 ± 0.010 (Fig. 2a), Z_p = 0.008 ± 0.001 (Fig. 2b), and Z_c = 0.815 ± 0.001 (Fig. 2c), which was approximately 3-fold, 2-fold, and 14-fold increase in the contents of ionic component, protein, and carbohydrate compared to untreated samples, respectively. This is because PEF (2–6 kV/cm) causes irreversible electroporation of the cell membrane, and HPH (100 MPa, 10 passes) results in the complete cell disruption and the formation of debris, improving the extraction process (Demir et al., 2023; Puértolas et al., 2016).

Fig. 2.

Extraction indexes of ionic components, Z_i (a), protein, Z_p (b), carbohydrate, Z_c (c), and total selenium, Z_Se (d) under pulsed electric fields (PEF) treatment (E = 0–6 kV/cm, t = 0–5.65 ms) and high pressure homogenization (HPH) treatment (p = 100 MPa, N = 0–10).

Note that for PEF treatment, the application of higher electric field strengths allowed facilitate the release of more ionic components and carbohydrates (Fig. 2a and c). However, the extraction of protein at higher electric field strengths (E = 6 kV/cm) was ineffective when compared with the results obtained at relatively lower electric field strengths (E = 2 kV/cm and 4 kV/cm) (Fig. 2b). This phenomenon could be attributed to the protein aggregation forming during PEF treatment. Applying a higher electric field strength can resulted in conformational changes in proteins, such as the exposure of hydrophobic regions or the formation of disulfide bonds. These changes promote protein-protein interactions, resulting in the formation of insoluble aggregates that reduce protein solubility and extractability (Wang et al., 2025). Another possible explanation for the decrease in protein content during PEF treatment at 6 kV/cm is protein degradation. High-energy PEF can cause localized heating or generate reactive species that can cleave peptide bonds, producing smaller peptides or fragments. These degradation products may not be fully quantifiable using conventional protein assays (e.g., the Bradford method) (Akaberi et al., 2019). Besides, the values of Z_i, Z_p, and Z_c roughly increased with PEF treatment time or HPH cycle number. The extractability of intracellular components for both PEF and HPH treatment can be ranked in the order of Z_i > Z_c > Z_p. Interestingly, the highest extraction index for carbohydrate (Z_c ≈ 0.8) was observed with PEF at 6 kV/cm and after treatment of 5.65 ms, while the highest extraction indexes for ionic component (Z_i ≈ 1) and soluble protein (Z_p ≈ 0.06) were obtained with HPH treatment after 10 passes. The lower protein release from electroporated KFP cells (PEF treatment) can be attributed to either to the pores formed being insufficiently large to release proteins with higher molecular weights, or to electrostatic interactions between intracellular proteins and the negatively charged phosphate groups in the mannan side chains. Additionally, the relatively low Z_p value observed in HPH-treated KFP cells in the present study may indicate that the intense interfacial shear stresses and inherent heating occurring in the homogenization valve induce degradation of certain proteins (Donsì et al., 2010; Thomas & Geer, 2011). These results, coupled with insights from other studies on cell disintegration caused by PEF and HPH (Carullo et al., 2018; Carullo et al., 2022; Marín-Sánchez et al., 2024b), suggest a difference in the mechanisms by which the two methods affect cells.

However, different extraction behavior was observed for total selenium (Fig. 2d). In contrast, the value of Z_Se increased with t and then decreased, reaching its maximum (Z_Se ≈ 0.56, 0.67, and 0.82 for 2 kV/cm, 4 kV/cm and 6 kV/cm, respectively) for PEF treatment at t = 1.13 ms. The observed changes of Z_Se were rather similar for HPH treatment, but they were more pronounced at N = 2. These phenomena can be explained by the extended treatment time resulting in excessive instantaneous temperature, and biological selenium is more sensitive to treatment temperature, generating volatile selenium and leading to the loss of selenium (Lewis et al., 1966). Besides, the PEF treatment process probably causes protein denaturation and hydrolysis by activating natural enzymes in KFP, resulting in binding Se speciation and content change (Pérez et al., 2018). PEF treatment can influence the activity of endogenous enzymes in two ways. Firstly, moderate-intensity electroporation of cell membranes (e.g. 2–4 kV/cm in this study) can enhance the accessibility of intracellular enzymes by disrupting membrane barriers, thereby increasing their contact with substrates (Marín-Sánchez et al., 2024a). Secondly, the effect on enzyme catalytic activity depends on PEF intensity and treatment duration. Mild PEF conditions generally preserve enzyme structure and may even induce conformational changes that increase catalytic efficiency. In contrast, higher field strengths or extended treatment times can cause partial unfolding or denaturation due to localized Joule heating or reactive oxygen species generation, leading to reduced enzyme activity (Marín-Sánchez et al., 2024b). In this study, PEF treatment at 2–4 kV/cm likely activated intracellular enzymes (e.g. proteases and selenocysteine lyases), accelerating the hydrolysis of Se-containing proteins (e.g., Se-cysteine-rich proteins). This explains the correlation between reduced the selenium content and decreased protein extractability. At 6 kV/cm, the slower increase in protein extractability (Fig. 2b) may indicate partial protease inactivation, limiting further protein hydrolysis and selenium release. This aligns with previous reports that enzyme activity under PEF is intensity-dependent, with optimal activity often being observed at moderate field strengths (Carullo et al., 2022). Thus, PEF-mediated changes in enzyme activity likely contribute to the dynamic release and transformation of intracellular components, including proteins and selenium. The data from the untreated samples indicate that in the absence of temperature variations, the total selenium content increases with prolonged extraction time (Fig. 2d).

Moreover, the protein purity and selenium content obtained after different treatments are detailed in Table 1. In comparison with untreated samples, the total selenium content and organic selenium content of the extract obtained after PEF treatments at E = 2–6 kV/cm for t = 5.65 ms, were significantly reduced by 6.0 % ∼ 37.9 % and 31.7 % ∼ 68.3 %, respectively (p < 0.05). The values of protein purity, total selenium, inorganic selenium and organic selenium contents, organic selenium ratio of the extract increased with the increase of E. The maximum values of protein purity and total selenium content were observed for HPH treatment, but a 15 % reduction in the organic selenium ratio compared with untreated sample. One possible explanation is that organic selenium may vaporize or volatilize during the PEF or HPH treatment process, transforming into inorganic selenium (Fu et al., 2018). The same phenomenon has also been observed, different treatments, such as boiling, steaming, and microwaving, led to varying degrees of selenium loss were reported (Pérez et al., 2018). Additionally, the obtained data evidenced that the total selenium and organic selenium contents correlated positively with protein (Table 1). This is because selenium exists in plant proteins mainly in the organic forms of Se-cysteine (Se-Cys) and Se-methionine (Se-Met), and is indirectly released through the dissolution of many proteins during cell disruption.

Table 1.

Protein purity and selenium content of aqueous extracts obtained by different methods.

Treatments	Protein purity (%)	Total selenium content, (CSet, mg/kg)	Inorganic selenium content (CSeI, mg/kg)	Organic selenium content (CSeo, mg/kg)	Organic selenium ratio (%)
Untreated (0 kV/cm)	21.323 ± 0.150e	0.248 ± 0.027ab	0.065 ± 0.006c	0.183 ± 0.007a	73.75 ± 2.05a
PEF (2 kV/cm, 5.65 ms)	24.287 ± 0.188d	0.154 ± 0.007c	0.096 ± 0.010b	0.058 ± 0.010e	37.63 ± 5.33c
PEF (4 kV/cm, 5.65 ms)	26.420 ± 0.364c	0.175 ± 0.002c	0.097 ± 0.002ab	0.078 ± 0.015d	44.74 ± 0.84c
PEF (6 kV/cm, 5.65 ms)	27.493 ± 0.281b	0.233 ± 0.007b	0.118 ± 0.020a	0.125 ± 0.009c	53.82 ± 3.83b
HPH (100 MPa, 10 passes)	31.277 ± 0.210a	0.270 ± 0.025a	0.113 ± 0.010ab	0.157 ± 0.010b	58.22 ± 3.15b

^a,b,c,d,e: different superscripts in each column mean a very significant difference (p < 0.05).

3.2. Effect of PEF treatment on extraction selectivity

The selectivity index, S = F_c/F_p, was further calculated for characterize the relative selectivity of carbohydrate and protein extraction, which was useful for estimating the purity of the extract (Zhang et al., 2020). For non-selective extraction, a value of S = 1 is expected (blue dashed line in Fig. 3). Fig. 3 illustrates that the selectivity index, S, versus specific energy consumption, W, for PEF treatment (E = 0–6 kV/cm, t = 0–5.65 ms) and HPH treatment. S values of ∼1.851 were observed for the untreated sample (0 kV/cm), while for both the PEF and HPH treatments, the S value exceeded 2. This suggests that the applied physical treatments had positive effects on the selective extraction of carbohydrates compared to proteins. During PEF treatment, the S value increased as the electric field strength increased. For example, PEF treatment for 5.6 ms at 2 kV/cm, 4 kV/cm and 6 kV/cm resulted in S = 2.720, 3.240, and 6.106, respectively. However, a higher S value was obtained by using a higher energy consumption (Fig. 3). The possible reason was that the higher energy consumption leads to a more effective electroporation process. This increased permeability and electroporation preferentially enhance the mass transfer rate of small molecule components such as ionic and carbohydrates (Carullo et al., 2018; Zhang et al., 2021). It was also found that the energy consumption of HPH was significantly higher than that of PEF. Despite this increase in energy consumption during HPH treatment, the selectivity index remains around S ≈ 2.5 after different numbers of passes and is not significantly increased. For example, at an equivalent energy consumption level (W ≈ 3 kJ/g DM), PEF treatment gave S ≈ 3.0 at 4 kV/cm and S ≈ 4.2 at 6 kV/cm, whereas HPH treatment gave S ≈ 2.3. Furthermore, at a selectivity index of approximately 2.5, HPH treatment requires a minimum energy consumption of 10 kJ/g DM, whereas PEF treatment requires approximately 1 kJ/g DM (4 kV/cm, 1.13 ms). This demonstrates that PEF allows for higher selectivity with lower energy consumption.

Fig. 3.

Selectivity index (S) versus specific energy consumption (W) for pulsed electric fields (PEF) treatment (E = 0–6 kV/cm, t = 0–5.65 ms, W = 0–8.36 kJ/g) and high pressure homogenization (HPH) treatment (p = 100 MPa, N = 10, W = 0–19.05 kJ/g). Here, S = F_c/F_p, F_c = C_c/C_c^m, F_p = C_p/C_p^m, where C represents the contents of carbohydrates and proteins during treatments, and C^m represents the maximum contents of carbohydrates and proteins.

3.3. Effect of PEF treatment on the particle size distribution

Fig. 4 presents PSD data for 5 % suspension treated by PEF treatment at different electric field strengths (t = 5.65 ms). The untreated suspension (0 kV/cm) shows an approximately normal distribution with a single peak at 163 μm, reflecting the size of the KFP cell or clusters. The appearance of cell clusters can be attributed to the small size of the KFP particles, which tend to bind to small insoluble particles and aggregate easily before complete stirring (Ning et al., 2020). The unimodal size distribution is still evident after the application of PEF treatment at 2 kV/cm. However, as the electric field strength increases, the peak gradually shifts towards larger particle sizes and the shape of the curve also slight changes. For E = 4 kV/cm and 6 kV/cm, the PSD revealed the presence of the bimodal distributions with peaks at ≈ 13 μm and 200 μm, corresponding to the formation of cell debris and cell conglomerates, respectively. The observed phenomenon, which is related to the mechanism of action of the PEF treatment on the particles in suspension, may lead to aggregation or dispersion of the particles, thereby affecting the particle size distribution (Zhang et al., 2019).

Fig. 4.

Particle size distributions (PSD) of both untreated and pulsed electric fields (PEF) treated konjac fly powder (KFP) suspensions.

3.4. Effect of PEF treatment on the cell microstructure

The microstructural changes in the KFP cells were further assessed using SEM. As shown in Fig. 5, the untreated KFP exhibited an intact cell structure, appearing nearly round, or polygonal in shape. After PEF treatment at 2 kV/cm, the surface becomes wrinkled with small interspaces or holes, and the polygonal shape becomes more pronounced (Fig. 5b). This is attributed to the transient pressure shock wave released externally by the high-pressure pulse during PEF treatment, which leads to the electroporation of the cell membrane and can create interspaces, holes and microfractures in cells, ultimately resulting in shrinkage (Delran et al., 2023; Gateau et al., 2021). After the application of PEF treatment at 4 kV/cm, the roughness of the cell surface is further increased, with some obvious depressions or bumps appearing, and the integrality of the cell shape is disrupted (Fig. 5c). When the PEF treatment is applied at 6 kV/cm, the cell breaks become more pronounced. There are more irregular structures on the surface and some cells even show signs of lysis, resulting in blurred cell boundaries (Fig. 5d). However, the wall structure was completely disintegrated after application of HPH treatment with exposure of the inner structure and release of the cellular contents (Fig. 5e).

Fig. 5.

Micrographs (a-e) and fractal dimension (FD) (f) of konjac fly powder (KFP) cells obtained from different treatments.

To further quantitatively assess the microstructural changes of KFP cells under different treatments, the SEM images were binarized and used for the FD calculation. The FD is commonly used to evaluate how an object, such as a particle, occupies space. A line has a FD of 1, a two-dimensional plane has a FD of 2, and a sphere will have a FD of 3. It is also affected by the complexity of the shape or surface roughness, making it useful for quantifying different degrees of agglomeration. More compact agglomerations, which have more complex surface structures, are likely to result in a higher FD (Langrish et al., 2006). The changes in FD of KFP cells under different treatments are shown in Fig. 5f. It was observed that the untreated cells had the highest FD value of 1.798. After PEF treatments, the FD value decreased from 1.773 to 1.671 as the electric field strength increased from 2 kV/cm to 6 kV/cm. This suggests that the cell morphological complexity of KFP cells gradually decreased as the electric field strength increased. The effect of microwave- and ultrasound-assisted extraction (MAE and UAE) on cell morphology of cabbage was investigated by the researchers (Pongmalai et al., 2015). They found that UAE, MAE and UAE + MAE caused significant changes in cell surface features, resulting in an increase in FD, which is opposite to our findings. This phenomenon is attributed to the unique characteristics of KFP. The KFP cells are tiny and when water is added they immediately agglomerate and become sticky, increasing the surface complexity. After treatment with PEF, these agglomerates begin to disperse to some extent. Fitting analysis of the data shows that the relationship between electric field strength and FD is as follows: FD = −0.0255 E + 1.8299 (R² = 0.9993). This reflects that there is a linear correlation between the electric field strength and the FD of KFP cells, and that the FD decreases linearly as the electric field strength increase. Moreover, the lowest FD value (1.640) was obtained after HPH treatment, which is due to the more intense mechanical forces and cavitation effects in HPH that cause more severe disruption and simplification of cell structure, resulting in a significant decrease in the FD of cells.

3.5. Correlation between cell disruption degree and intracellular components extractability

The release efficiency of small molecules, such as ionic components, can be evaluated by the cell membrane permeability, whereas the efficient release of components with higher molecular weights or lower solubility in water could require more intensive cell walls damage. In this study, the PEF treatment medium was a 5 % KFP suspension, prepared using deionized water with an initial conductivity of ∼1500 μS/cm. The electrical conductivity and dielectric constant of the medium are crucial in determining the electric field distribution, energy dissipation extent, and ultimately membrane electroporation intensity (Saulis, 2010). A medium with higher conductivity facilitates current flow, leading to faster energy transfer to the cell membrane. However, it may also increase Joule heating, which can affect membrane resealing and structural integrity (Guo et al., 2023). Conversely, a medium with lower conductivity can reduce heating, but may require a higher field strength to achieve comparable electroporation effects (Ivorra et al., 2010). In our experiments, conductivity and temperature were kept constant by using an ice-water bath to ensure that observed membrane disruption and extraction efficiencies were primarily due to the electric field strength rather than uncontrolled medium variations. In this line, the correlation between extractability of intracellular compounds and cell disruption degree induced by PEF (0–6 kV/cm) was further evaluated in Fig. 6. Compared with the untreated sample, PEF treatment allowed significantly improved the degree of cell disruption, Z_d, and that the value of Z_d increased with the increasing electric field strengths. It was also observed that there exists a complicated correlation between the different extraction indexes of intracellular components and the cell disruption index. These dependencies exhibited significant non-linearity. Z_i shows a general positive correlation with Z_d, meaning that the cell membrane permeability (i.e. Z_i) increases with the degree of cell disruption (i.e. Z_d) increases. At small values of Z_d < 0.5, the increase in Z_i is relatively slow. This suggests that although the integrity of the cell wall was partially damaged by the low intensity PEF treatment, the relative stability of the sample tissue and cell membrane can be maintained due to the self-regulatory and protective mechanisms of the cell, so that the increase in Z_i was relatively slow. At higher levels of cell disruption (Z_d > 0.5), Z_i shows a significantly greater rate of increase, indicating that when cell wall damage reaches a certain level, the protective effect of the cell wall on the sample tissue and cell membrane is almost completely lost, making them more susceptible to direct external influences, leading to a rapid escalation of cell membrane permeability. A similar extraction behavior was observed for Z_c at Z_d < 0.5. This also reflects that a low-intensity or early-stage PEF treatment induced the cell membrane permeability increase and carbohydrate release in almost near linear dependencies. Subsequently, as the degree of cell disruption increased (Z_d > 0.5), the rate of carbohydrate release also significantly increased. Interestingly, the extraction indexes of protein and total selenium showed different trends at different stages of cell disruption (Z_d < 0.2; 0.2 < Z_d < 0.5; Z_d > 0.5), and these changes may be influenced by a combination of factors such as the degree of cell disruption, changes in the intracellular environment, and the forms of protein and selenium present and their interactions with other substances in the presence of PEF (Fu et al., 2018). Therefore, the observed different behavior of intracellular components extraction indexes reflected different effects of PEF on disruption of cell membranes (so-called electroporation) and cell walls.

Fig. 6.

Correlation between extraction indexes of ionic components, Z_i, protein, Z_p, carbohydrate, Z_c, and total selenium, Z_Se and cell disruption index, Z_d, for pulsed electric fields (PEF) treated konjac fly powder (KFP) cells.

4. Conclusions

In this study, the effect of PEF-assisted enhancement of cell disruption and selective recovery of intracellular components from selenium-enriched KFP was investigated and compared with HPH treatment. The correlation between the extractability of intracellular compounds (Z_i, Z_p, Z_c, Z_Se) and the degree of cell disruption (Z_d) caused by PEF was evaluated for the first time. The results showed that PEF treatment significantly improved the degree of cell disruption and the extractability of intracellular components. For example, PEF treatment at 6 kV/cm for 5.65 ms (Z_d = 0.86) resulted in approximately 3-, 2-, and 14-folds higher contents of ionic component, protein, and carbohydrate compared to untreated samples ((Z_d = 0), respectively. However, PEF was less effective for extracting protein than carbohydrate, requiring more intensive techniques such as HPH or ultrasonication treatment. Furthermore, the observed nonlinear dependencies of Z_i, Z_p, Z_c, Z_Se and Z_d reflected the different effects of PEF on the disruption of cell membranes (electroporation) and cell walls. Although both PEF and HPH are scalable, further studies are needed to optimize the conditions, evaluate the impact of extracting and fractionating multiple intracellular components simultaneously, and characterize their functional and nutritional properties for use in the food industry.

CRediT authorship contribution statement

Xun Pei: Writing – original draft, Investigation. Jinzeng Wang: Formal analysis. Chen Liu: Writing – original draft, Formal analysis. Zushan Tan: Investigation, Data curation. Yilin Li: Investigation, Data curation. Muci Wu: Writing – review & editing. Wangting Zhou: Writing – review & editing. Jingren He: Writing – review & editing. Nabil Grimi: Writing – review & editing. Rui Zhang: Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.