DOI: https://doi.org/10.21203/rs.3.rs-2746520/v1
To reduce the adverse physical effects on the oral mucosa caused by excessive hardness of betel nut fibres, steam explosion was used to soften betel nuts. The effect of 3 operating parameters (pressure holding time, explosion pressure and initial moisture content) on the morphology, texture and chemical composition of the betel nuts was investigated. The fibre hardness and Shore hardness decreased by 56.17%-89.28% and 7.03%-34.29%, respectively, and the transverse tensile strength and fibre tensile strength also decreased by up to 60.72% and 24.62%, respectively. Moreover, the coefficient of friction and moisture content increased. After steam explosion, the betel nut increased in transverse diameter, became darker and more yellow‒red in colour, and showed a damaged microstructure. The contents of lignin, hemicellulose, free phenol, bound phenol and alkaloids decreased after steam treatment, with total phenols and alkaloids decreasing from 44.07 mg(GAE)/g and 7.84 mg/g to 30.61 mg(GAE)/g and 6.50 mg/g, respectively, after the A-50 s treatment condition. A slight increase in cellulose was observed when steam explosion conditions exceeded 80 s or 0.75 MPa. The steam explosion increased the quantity of phenols, alkaloids and soluble solids released from the betel nut under the same simulated release conditions. The research also showed that the explosion efficiency was positively correlated with the pressure holding time and explosion pressure, while the initial moisture content was reduced the explosion efficiency. Therefore, steam explosion is an effective pretreatment approach to soften betel nut and facilitate healthy development of the betel nut industry.
Betel nut is widely cultivated as a cash crop in Southeast Asia, South Asia and Hainan, China(Chen et al., 2021). Betel nut contains alkaloids, polyphenols and other active substances. As a traditional medicine in China and India, betel nut can help with resistance to bacteria, removal of parasites and facilitation of digestion(Yi et al., 2022); on the other hand, as an addictive drug and chewable products, the physical action of the betel nut fibre can puncture the oral mucosa, and excessive long-term chewing can lead to oral inflammation and fibrosis(Gupta et al., 2020). Fresh betel nuts are typically chewed with slaked lime and Laotian leaves or grated tobacco(Dalisay et al., 2019). In China, dried betel nut is typically packaged into commercial products by boiling, adding flavour, stewing until it is fragrant, adding brine, and drying. As a result, the effective softening of betel nut fibres to improve quality has become a focal point for the healthy development of the betel nut industry.
Steam explosion is an effective pretreatment approach often used in the modification of plant fibres, extraction of substances, etc.(Ma et al., 2022). The process uses steam as a medium to maintain an environment of high temperature and pressure for a period of time and then quickly releases the system to atmospheric pressure, converting thermal energy into mechanical energy(Yu et al., 2022). Commonly used methods of fibre softening can be divided into physical, chemical, biological, or combined methods(Rajan et al., 2022). However, chemical residues, byproducts, loss of active ingredients, and high cost limit the application of softening in betel nut processing(Akhila et al., 2005). During steam explosion, the high temperature, high pressure, and high humidity, combined with the explosive effect of steam expansion, leads to degradation and mechanical fracture of the material, which can cause the fibres to become soft and loose(Wi et al., 2013). As a result, steam explosion can be used as a pretreatment method to soften the fibres and improve the quality of betel nut processing. Many studies have reported on the use of steam explosion in lignocellulosic materials. For example, Shu et al.(2021) designed a single-factor experiment of steam explosion pressure and holding time to explore the effect of steam explosion on moso bamboo and poplar fibre. Kong et al.(2022) studied the effect of different steam explosion conditions on the morphology and chemical composition of soybean meal. However, the use of steam explosion for food texture modification is rarely reported. Therefore, it is necessary to explore the influence of different steam explosion conditions on the softening of betel nut.
The present study investigated the effects of steam explosion with different explosion pressures, pressure holding times, and initial moisture contents of betel nut on the softening effect of the fibres as well as the physical properties, appearance, and chemical compositions. This study provides a theoretical basis for better application of steam explosion approaches to betel nut fibres and to reduce the detrimental effects of excessive fibre hardness.
Dried betel nut was supplied by Hainan HoChung Food Technology Co., Ltd. (Dingan, China). DNS reagent, Folin-phenol, and artificial saliva were purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China). Arecoline, Arecaine, Guvacoline, and Guvacine were purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, China). All other chemicals and solvents were analytical grade. The pure water used in this study was produced using the Urifier ultra-pure water system (Shanghai, China).
The steam explosion was performed on the QBS-80 steam explosion machine (Henan Zhengdao Biological Energy Co., Ltd., China), which consisted primarily of a steam generator and a reactor chamber. Based on the actual working experience, the steam explosion pressures used in the tests were 0.15, 0.25, 0.35, 0.55, 0.75 and 0.95 MPa for 50 s (Group A); the pressure holding times were 0, 5, 20, 50, 80 and 110 s at 0.35 MPa (Group B); the initial moisture content was controlled by soaking for 0, 2, 5, 8, and 24 h (Group C), the remaining samples require no soaking. After the steam explosion, the samples were collected and cooled to room temperature, enclosed and stored at -20℃.
All samples were randomly selected and placed in a clean petri dish, and photographs were recorded. The transverse diameters of each sample were measured using a Vernier calliper with a precision of 0.02 mm(Wang et al., 2021). Testing on each sample was repeated 16 times.
A WSC-1B colorimeter (Shanghai Yidian Physical and Optical Instruments Co., Ltd., China) was used to measure the colour of over 40 mesh betel nut powders. The instrument was calibrated using black and white tiles. The values L*, a* and b* were recorded. The process was repeated five times for each sample. L* reflects the luminance of the colour, with 0 representing black, and 100 representing white; a* reflects the concentration of red or green: a > 0 indicates reddish, a < 0 indicates greenish; b* reflects the concentration of yellow or blue: b > 0 indicates yellowish, b < 0 indicates blueish(Gong et al., 2021).
The moisture content of betel nut was tested by the direct drying method(Chen et al., 2022). The initial weight (W1) of the randomly selected whole betel nuts was determined and recorded using a PL3002 electronic weighing balance (METTLER TOLEDO Group, Swiss) with a sensitivity of 0.01 g. Then the samples were put in a DHG-9140 Electric Heating Dryer (Shanghai Yiheng Scientific Instruments Co., Ltd., China) at 105℃ and dried to constant weight (W2); prior to each weighing, the sample was cooled in a desiccator. The moisture content was determined by the relation (W1-W2)/W1. Testing on each sample was repeated 5 times.
Small pieces (2 × 1 cm) were cut from the middle of the betel nut and held in an FAA fixative solution (a 1:1:18 ratio of formaldehyde, glacial acetic acid and ethanol by volume) at 4 ℃ for 24 h. The specimens were then cut into thin slices with a sterilized knife(Chen et al., 2021). Slices were stained using a 1% (W/V) safranin solution for 0.5 h and washed with deionized water three times. Slices were then dehydrated and decolorized through a graded ethanol series (60, 70, 80, 90, and 100%) for 2 min each. Finally, the samples were cleared with a transparent solution [glycerol, gelatine and water (12 mL: 1 g: 100 mL)] and observed under an AE31 light microscope (MOTIC China Group Co., China)(Lian et al., 2020).
The microscopic morphology of the betel nut surface, cross-section, and longitudinal section at 100x and 3000x magnification were observed and photographed using a scanning electron microscope (Hitachi, Ltd., Japan). Prior to observation, samples were air-dried and cut, mounted on a stub with conductive adhesive tape, and sputter coated with gold in an ISC-150 ion sputter coater (Shenzhen Supu Instrument Co., LTD, China)(Zhu et al., 2022).
Referring to the methods of Wetchakama et al.(2019) (with a few modifications), the fibre hardness of the betel nut was determined using a three-point bend method on a TA XT plus C texture analyser (Stable Micro Systems, United Kingdom) equipped with an HDP-3PB probe. A force‒time curve was generated from the compression. The largest force value was taken as the measure of hardness. A longitudinal betel nut piece (1 × 2 cm) was placed on the two base supports at a distance (span) of 1 cm, with the epidermis facing upward. The test conditions set for the instrument were a pretest speed of 3.00 mm/s, a test speed of 1.00 mm/s, a posttest speed of 3.00 mm/s, a trigger force of 20.0 g and a deformation of 40%. Testing for each sample was repeated 20 times.
A betel nut was split into two parts and placed on the loading platform, with the epidermis facing upward. The Shore hardness of the betel nut was determined using an XHS Shore A hardness tester (Yingkou Material Testing Machine Co., Ltd., China) with a press pin diameter of 0.79 mm and a load of 750 g. The Shore hardness is inversely proportional to the depth of the pointer in the middle of the betel nut(Esteves et al., 2021). Testing for each sample was repeated 20 times.
A texture analyser equipped with an A-ATG probe was used to determine the transverse tensile strength of the betel nut(Liu et al., 2022). The betel nut was cut into 0.25 cm wide transverse strips that were clamped at both ends (by the top and bottom probes) for stretching. The largest force value was recorded as the transverse tensile strength in a force‒time curve. The test conditions for the instrument were a pretest speed of 5.00 mm/s, a test speed of 2.00 mm/s, a posttest speed of 5.00 mm/s, a trigger force of 5.0 g and a displacement of 20 mm. Testing for each sample was repeated 12 times.
The typical inclined slope method was used to determine the coefficient of static friction of the betel nut surface. The apparatus comprised two smooth wooden planks and a protractor. The two planks were connected by hinges and able to be freely opened and closed, and the protractor was attached to a single plank to measure the angle between the planks(Zhang et al., 2022). The betel nut was laid on a plank, as shown in Fig. 1. The plank slowly tilted until the movement of the betel nut down the slope began. The angle was then read from a protractor, and the tangent of the angle represents the coefficient of static friction. Testing of each sample was repeated 20 times.
The dried betel nut was cut and soaked in distilled water for 1 week. Then, the samples were held in 8% NaOH (w/v) for 24 h and 5 g/L sulfuric acid for 1 h. The fibres were finally pounded for loosening, washed in running water and dried(Srinivasa et al., 2011). Single fibres were gently peeled, trimmed to 1.5 cm, and weighed for calculation of linear density (mg/cm). To prevent slipping, cut ends were dipped in epoxy resin (epoxy resin: hardener 3:1). The ends of the epoxy-wrapped fibres were clamped using the air-clamp of the 3343 Universal Material Testing Machine (Instron Limited, USA)(Yusoff et al., 2016). The test conditions set for the instrument were a pretest speed of 0.5 mm/s, a test speed of 0.2 mm/s, and a posttest speed of 0.5 mm/s and the remainder use default settings. The tensile strength was obtained from the load displacement curves recorded during the tensile tests. Due to differences in fibre diameters, the load was divided by the linear density to compare the load values per unit linear density. Testing for each sample was repeated 10 times.
The betel nut samples were crushed into powder, passed through a 40-mesh sieve and dried to a constant weight. The content of acid-insoluble lignin was determined by the concentrated sulfuric acid method. The hemicellulose content was determined by the hydrochloric acid-DNS reducing sugar method. Detailed steps were described by Wu et al.(2021). The cellulose content was determined by the nitric acid-ethanol method(Yang et al., 2021). Briefly, samples (0.75 g) were added to 25 mL of nitric acid-ethanol (4:1, v/v), heated for 1 h in a boiling water bath with stirring every 10 min, centrifuged at 6000 rpm for 5 min at 4 ℃ on an H1850R centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., China) to remove the supernatant fluid, and repeated once. Finally, a large number of insoluble residues were obtained by filtration, and the weight was the weight of cellulose after drying. Testing for each sample was repeated 10 times.
Free polyphenols were extracted using ethanol assisted with ultrasound(Guerrini et al., 2020). Two grams of betel nut powder over 40 mesh sizes was mixed with 30 mL of 70% ethanol (v/v) and placed in a SB-4200DTS ultrasonic generator (400 W, 40 kHz) (Ningbo Xinzhi Biotechnology Co., Ltd., China) for 30 min. The mixture was then heated at 60°C in a water bath for 1 h and centrifuged at 6000 rpm for 5 min at 4℃ on an H1850R centrifuge. After two extractions, the supernatant was combined and then diluted 25 times as the solution to be tested. Testing for each sample was repeated 5 times.
Bound phenols were extracted after removing the free polyphenols, with the remaining material washed with distilled water, dried, and then mixed with 2.5 mol/L NaOH at a ratio of 1:15 (w/v) for 10 h in a SHA-2 thermostatic shaker (Changzhou Aohua Instruments Co., Ltd., China). The pH of the hydrolysed product was adjusted to 1.5–2.5 by 6 mol/L hydrochloric acid, and the product was extracted five times with an equal volume of ethyl acetate. The supernatants were combined and dried using a RE501 rotary evaporator (Shanghai Yuanhuai Industrial Co., China) under a vacuum at 40 ℃. The dried residue was finally redissolved with 50% methanol (v/v) to 10 mL and diluted 10 times as the test solution(Li et al., 2022). Testing for each sample was repeated 5 times.
The total polyphenol content of the extract was determined using the Folin-Phenol method(Li et al., 2022). A standard curve was prepared using a gallic acid series from 10 to 50 µg/mL for testing. A total of 1.0 mL from each test solution was mixed with 5.0 mL of 10% Folin-Phenol solution, reacted for 5 min, and then 4.0 mL of 7.5% Na2CO3 solution was added to the reaction at room temperature for 1 h in the dark. The absorbance was measured at 765 nm using a synergy lx microplate reader (Biotek Instruments, Inc., USA). The results were calculated with the standard curve and were expressed as mg of gallic acid equivalents (GAE) per 1 g dry weight of betel nut (mg GAE/g).
Extraction of alkaloids involved adding the betel nut powder with grain size over 40 mesh to 80% ethanol solution (v/v) according to the material-liquid ratio of 1:25 and sonicating the mix (400 W 40 kHz) for 30 min. The mixture was then heated in a water bath at 80℃ for 1 h and centrifuged at 6000 rpm for 5 min at 4℃. After two extractions, the supernatant was combined, diluted 4 times and filtered through 0.22 µm nylon micropore membranes as the solution to be tested(Musdja & Musir, 2020). Testing for each sample was repeated 5 times.
Alkaloid determination considered four betel nut alkaloids (Arecoline, Arecaine, Guvacoline, Guvacine) using a High-Performance Liquid Chromatograph (1260, Agilent Technologies, USA) on an SCX column (250 mm × 4.6 mm, 5 µm) (Agilent Technologies, USA) at 30℃ by DAD detection at 215 nm(Petruczynik et al., 2018). The mobile phase was a phosphoric acid (0.1%) solution (A) and acetonitrile (B) at a flow rate of 1.0 mL/min (A:B, 7:3). The mobile phase was filtered through a 0.22 µm nylon filter before use.
This method is a modification of previous methods for simulate chewing using a texture analyser(Kim et al., 2019; Liu et al., 2020). A betel nut was cut in half and soaked for 16 h at in volume of 10 mL per half of the betel nut, after which it was placed in a 60 mm petri dish and selected for the texture analysis P/36r column probe. The test conditions set for the instrument were a pretest speed of 40 mm/s, a test speed of 40 mm/s, a posttest speed of 40 mm/s and a trigger force of 5.0 g. The betel nut was pressed 120 times at 60%, 75%, 90%, 90%, and 90% deformation for a total of 600 times. Five millilitres of artificial acidic saliva at 37°C was replaced every 120 presses, and the betel nut was turned over constantly during this time. The collected liquor was determined for phenols and alkalis according to previous methods, and the soluble solids were determined using a PAL-1 digital refractometer (ATAGO Scientific Instruments Co., Japan). Testing for each sample was repeated 6 times.
The results are expressed as the mean ± SD. Statistical analyses were performed using IBM SPSS Statistics 26 software (IBM Corporation, USA). The differences between groups were distinguished by one-way ANOVA and Duncan’s multiple range tests, and P <0.05 was considered statistically significant and indicated by different letters. Some data were plotted using Origin2022b software (OriginLab Corporation, USA).
3.1 Effect of different steam explosion conditions on the macroscopic morphology and moisture content of betel nut
The appearance of a food product is an important quality that determines first impression. This is also true for betel nut as a local specialty and chewable product. The results of the transverse diameter and macroscopic morphology of betel nut are shown in Table 1 and Fig. 2. Untreated betel nut was a brighter green, with dense surface folds and tighter fibres. After the steam explosion treatment, the surface folds spread and changed from bright to dark green and showed a slightly orange‒red colour at both ends. When the pressure was 0.55 MPa, the surface folds nearly disappeared. When it reached 0.95 MPa, cracks appeared on the surface, the appearance was damaged, and it was no longer suitable for subsequent processing. A comparison of the cross-section of a betel nut shows that the internal fibres change from white‒green to brown after steam explosion. Decreasing the pressure or increasing the initial moisture content caused the appearance to more closely resemble the untreated betel nut.
The transverse diameter of the betel nut increased significantly with increasing pressure holding time and explosion pressure. The transverse diameter increased by 23.47% and 22.96% after the A-110 s and B-0.95 MPa treatments, respectively. The transverse diameter of the sample steam explosion after soaking was between that of the unsoaked and untreated samples. However, the transverse diameter of the samples did not change as the initial moisture content increased further. Whether due to the moisture in the steam or the moisture added by soaking, the betel nut fibre swells and its volume increases. Moreover, the pressure drops rapidly to atmospheric pressure when steam explodes, and the betel nut expands with the mechanical force generated by this gas expansion. Prolonging the pressure holding time and increasing the explosion pressure promotes further steam intrusion, and an increased initial moisture content can hinder high-pressure steam entry and steam release(Hong et al., 2020).
Colours are the synthetic result of the reflection of visible light by an object, as determined by the substance's chromophores and auxiliary chromophores. The colour of betel nut is mainly determined by pigments such as chlorophyll and lutein in the epidermis as well as lignin, which includes a large number of auxiliary chromophores and chromophores, as well as a few small but essential phenolic compounds and quinones and their oxides(Yazaki, 2015). These substances cause colour change as the steam explosion conditions change. According to the results of the different steam explosion conditions shown in Table 1, The L* value was negatively correlated with both the pressure holding time and the explosion pressure. There was a significant increase in the a* value compared to the untreated betel nut, indicating a reddish colour. A-50 s treatment increased from 12.81 to 16.02, and the a* value increased with pressure holding time and explosion pressure and decreased with increasing initial moisture content. For the b* value, there was a significant increase with increasing moisture content and pressure holding time, generally towards yellow. However, increasing or decreasing the pressure had little effect on the b* value. These findings are consistent with the reports of Gong et al.(2021).
| Samples | Transverse diameter (cm) | Moisture content (%) | L* | a* | b* |
|---|---|---|---|---|---|
| BN | 1.96 ± 0.18d | 8.51 ± 1.13%b | 64.72 ± 1.40a | 12.81 ± 0.85c | 40.78 ± 0.38c |
| A-0s | 2.21 ± 0.17c | 11.92 ± 0.38%a | 59.51 ± 0.22b | 15.98 ± 0.51ab | 41.08 ± 1.21c |
| A-5s | 2.23 ± 0.14bc | 12.93 ± 1.12%a | 60.27 ± 0.91b | 15.67 ± 0.09b | 40.45 ± 0.39c |
| A-20s | 2.25 ± 0.20bc | 12.75 ± 0.81%a | 60.58 ± 0.87b | 16.07 ± 0.40ab | 40.67 ± 0.25c |
| A-50s | 2.28 ± 0.17bc | 12.34 ± 1.57%a | 57.95 ± 0.79c | 16.02 ± 0.13ab | 42.33 ± 0.75b |
| A-80s | 2.32 ± 0.14ab | 11.95 ± 1.23%a | 57.19 ± 0.28c | 15.99 ± 0.33ab | 42.33 ± 0.26b |
| A-110s | 2.42 ± 0.22a | 13.86 ± 0.65%a | 57.41 ± 0.83c | 16.60 ± 0.07a | 44.31 ± 0.53a |
| BN | 1.96 ± 0.18e | 8.51 ± 1.13%d | 64.72 ± 1.40a | 12.81 ± 0.85d | 40.78 ± 0.38bc |
| B-0.15MPa | 2.17 ± 0.18d | 13.18 ± 2.18%bc | 65.10 ± 0.58a | 12.82 ± 0.40d | 41.22 ± 0.82b |
| B-0.25MPa | 2.25 ± 0.14cd | 12.35 ± 0.83%c | 59.22 ± 0.54bc | 16.19 ± 0.13b | 42.56 ± 0.31a |
| B-0.35MPa | 2.28 ± 0.17bcd | 12.34 ± 1.57%c | 57.95 ± 0.79cd | 16.02 ± 0.13b | 42.33 ± 0.75a |
| B-0.55MPa | 2.32 ± 0.16abc | 12.72 ± 1.16%c | 59.10 ± 1.05bc | 16.77 ± 0.39a | 39.93 ± 0.48c |
| B-0.75MPa | 2.40 ± 0.17ab | 14.86 ± 1.10%b | 60.00 ± 0.76b | 15.83 ± 0.11bc | 40.10 ± 0.62c |
| B-0.95MPa | 2.41 ± 0.15a | 17.65 ± 1.75%a | 55.86 ± 1.21d | 15.49 ± 0.04c | 38.88 ± 0.24d |
| BN | 1.96 ± 0.18c | 8.51 ± 1.13%f | 64.72 ± 1.40a | 12.81 ± 0.85d | 40.78 ± 0.38c |
| C-0h | 2.28 ± 0.17a | 12.34 ± 1.57%e | 57.95 ± 0.79cd | 16.02 ± 0.13a | 42.33 ± 0.75b |
| C-2h | 2.14 ± 0.13b | 18.49 ± 2.38%d (20.08 ± 0.88%) | 56.50 ± 0.94d | 16.04 ± 0.10a | 41.88 ± 0.60bc |
| C-5h | 2.11 ± 0.13b | 21.87 ± 2.30%c (28.07 ± 1.88%) | 61.98 ± 0.81b | 14.38 ± 0.49bc | 42.58 ± 0.46ab |
| C-8h | 2.14 ± 0.16b | 25.60 ± 0.67%b (32.15 ± 0.48%) | 59.96 ± 0.39c | 14.95 ± 0.08b | 42.66 ± 1.32ab |
| C-24h | 2.10 ± 0.12b | 30.53 ± 1.51%a (46.53 ± 2.03%) | 59.68 ± 0.72c | 13.92 ± 0.41c | 43.74 ± 0.66a |
| * The parentheses after group C represent the moisture content after soaking. (A-50 s, B-0.35 MPa, and C-0 h are samples with the same treatment conditions.) | |||||
The moisture content (Table 1) indicated a significant increase in moisture in most of the samples after the steam explosion and a significant increase with increasing explosion pressure, excluding the samples that had absorbed additional moisture due to soaking. This finding was also reported by Elliston et al.(2015). The moisture content reached 17.65% after the B-0.95 MPa treatment, which was 2.07 times greater than that before treatment. However, there was no significant difference in moisture content with increasing pressure holding time. For samples with increased initial moisture content through soaking, a significant moisture decrease was observed after steam explosion. After soaking for 24 h, the betel nut had a moisture content of 46.53%, which became 30.53% after the steam explosion. However, the moisture content of C-24 h was 3.59 and 2.47 times higher than that of the unprocessed and unsoaked samples, respectively. We propose these results are due to the adhesion of steam to the betel nut surface after steam explosion. As the explosion pressure increases, the fibres loosen and can absorb more water. However, the high temperature causes the original water to evaporate, and the reduced water holding capacity of the fibres after steam explosion leads to a loss of excess water after soaking(Zhu et al., 2022).
Optical and scanning electron microscopy observations showed significant modifications to the betel nut micromorphology under different steam explosion conditions. All of these modifications are attributed to the effects of high temperature, high pressure, and high humidity and the mechanical damage generated by steam explosion. As seen in the cross-section of the betel nut under optical microscopy in Fig. 3, the betel nut fibres before steam explosion were densely connected; after steam explosion, voids existed within and between the betel nut fibres; the epidermis was everted or burst and fell off. This variation shows a positive correlation with explosion strength. As shown in the betel nut under scanning electron microscopy in Fig. 3, the surface was compact, and the surface folds were densely intact under magnification. After steam explosion, the surface showed various cracks. With magnification, the grooves on the surface showed varying degrees of destruction and even disappeared completely, and holes and fragments appeared. The increased initial moisture content and reduced explosion pressure microscopic morphology of betel nut was closer to that of untreated betel nut. Similar changes in the microscopic morphology of steam explosion-treated soybean hulls were described by Zhu et al.(2022).
3.3 Effect of different steam explosion conditions on the texture and fibre tensile strength of betel nut
The texture of betel nuts directly affects their quality and is a judgement indicator of softening effectiveness. To compare the effects of the different steam explosion conditions on the texture and fibres of betel nuts, the results of betel nut fibre hardness, Shore hardness, transverse tensile strength, coefficient of friction and fibre tensile strength are shown in Table 2. All betel fibre hardness values significantly decreased after the steam explosion treatment. After the A-50 s treatment, hardness decreased by 61.20% from 10018.14 g to 3886.83 g. While the fibre hardness values decreased significantly with increasing explosion pressure and initial moisture content, there was no significant change in the effect on fibre hardness with increasing pressure holding time. This trend was also evident in the results for Shore hardness. However, the overall decrease ratio was not as great as that of the fibre hardness. The C-5 h treatment decreased the most, from 90.05 to 68.00, by 43%. A significant decrease in transverse tensile strength was observed with increasing pressure holding time, explosion pressure, and initial moisture content. Tensile strength decreased from 603.12 g to 449.06 g after A-50s treatment, a 25.54% decrease. A proposed explanation for this result is that when the steam explosion takes place, high-pressure steam is instantly released at atmospheric pressure, gas expands, and thermal energy is converted into mechanical energy to cause the explosion effect; this causes cracks and numerous small gaps to appear on the surface of the betel nut and internal fibres, reducing the strength of the interconnected fibre network(Tu et al., 2019). This phenomenon was also supported by observing the microstructure of the betel nut. Moreover, high temperature, high pressure and high humidity caused lignin, hemicellulose and a small amount of pectin between the fibres to degrade, broke hydrogen bonds, and resulted in inter-fibre bonds loosening. The increased moisture of samples also caused betel nut fibre softening, reducing the mechanical bond strength(Chang et al., 2012).
The static friction coefficient of betel nut is related to its texture, frictional damage. The typical inclined slope method is designed according to Newton's motion theorem, which is based on the fact that the component forces of gravity and friction along an inclined plane are equal when the object is about to slide; hence, µ = tanα. The results of the coefficient of friction on the betel nut surface showed a certain increase after steam explosion, and all betel nuts showed coefficients of friction greater than that between the experimental planks of wood (0.461 ± 0.042). In addition, the coefficient of friction increased significantly with increasing pressure holding time, explosion pressure and initial moisture content. After the C-24 h treatment, the coefficient of friction increased by 47.37% from 0.513 to 0.756. Based on previous studies, the coefficient of friction has been proven to have a positive correlation with moisture content(Fu et al., 2021). Thus, we presume that one reason for the increase in the coefficient of friction is the increase in moisture, which is supported by a similar trend in the results of moisture measurement. Another possible explanation is that the steam explosion treatment leads the otherwise smooth and firm epidermis to become rough and loose.
The betel nut fibre tensile strength per unit linear density fracture decreased after steam explosion treatment. However, the differences upon changing the steam explosion conditions were not statistically significant. After the A-50 s treatment, the fibre tensile strength decreased by 17.64% from 23.19 N to 19.10 N. This finding is consistent with Kim et al.(Kim & Fujii, 2009), who found a comparable reduction in tensile strength of bamboo fibres treated by steam explosion. These changes are attributed to the thermal degradation of cellulose as a result of the steam explosion process's high temperature, pressure and humidity as well as mechanical damage to the fibres due to gas expansion during pressure release. However, excess water blocks steam entry during the explosion process, and water's high specific heat capacity causes temperature to rise slowly, reducing the overall effect of the steam explosion(Sun et al., 2015).
| Samples | Fibre hardness (g) | Shore hardness | Transverse stretching (g) | Coefficient of static friction | Fibre tensile strength (N) |
|---|---|---|---|---|---|
| BN | 10018.14 ± 3879.62a | 90.05 ± 7.12a | 603.12 ± 110.80a | 0.513 ± 0.039b | 23.19 ± 3.92a |
| A-0s | 3831.94 ± 1042.93b | 82.77 ± 5.65b | 480.57 ± 77.89ab | 0.578 ± 0.068ab | 20.83 ± 3.58ab |
| A-5s | 4127.60 ± 1219.61b | 81.50 ± 7.51b | 474.80 ± 81.43ab | 0.555 ± 0.106ab | 18.63 ± 2.02b |
| A-20s | 3672.67 ± 971.41b | 81.82 ± 7.09b | 484.20 ± 78.78ab | 0.570 ± 0.060ab | 18.01 ± 2.65b |
| A-50s | 3886.83 ± 843.82b | 81.97 ± 5.83b | 449.06 ± 52.99ab | 0.559 ± 0.076ab | 19.10 ± 3.47b |
| A-80s | 3683.25 ± 976.67b | 78.94 ± 6.32bc | 415.4 ± 76.71b | 0.572 ± 0.083ab | 18.91 ± 1.71b |
| A-110s | 3620.66 ± 1384.99b | 75.17 ± 7.58c | 404.88 ± 124.12b | 0.619 ± 0.084a | 18.65 ± 1.65b |
| BN | 10018.14 ± 3879.62a | 90.05 ± 7.12a | 603.12 ± 110.80a | 0.513 ± 0.039b | 23.19 ± 3.92a |
| B-0.15MPa | 4390.89 ± 1787.47b | 81.27 ± 6.00bc | 491.84 ± 142.53ab | 0.567 ± 0.063ab | 19.64 ± 3.70ab |
| B-0.25MPa | 3980.25 ± 1161.67b | 83.72 ± 7.70b | 412.00 ± 66.18ab | 0.563 ± 0.093ab | 19.10 ± 3.47b |
| B-0.35MPa | 3886.83 ± 843.82b | 81.97 ± 5.83bc | 425.60 ± 81.55ab | 0.559 ± 0.076ab | 19.06 ± 2.53b |
| B-0.55MPa | 3816.54 ± 1578.37b | 79.67 ± 7.50bcd | 374.3 ± 77.14b | 0.547 ± 0.072ab | 17.89 ± 3.64b |
| B-0.75MPa | 2129.83 ± 613.54c | 75.00 ± 8.88d | 388.4 ± 67.30b | 0.602 ± 0.088a | 18.02 ± 2.28b |
| B-0.95MPa | 1767.90 ± 512.41c | 77.35 ± 7.67cd | 349.6 ± 61.24b | 0.602 ± 0.084a | 17.48 ± 2.47b |
| BN | 10018.14 ± 3879.62a | 90.05 ± 7.12a | 603.12 ± 110.80a | 0.513 ± 0.039d | 23.19 ± 3.92a |
| C-0h | 3886.83 ± 843.82b | 81.97 ± 5.83b | 449.06 ± 52.99ab | 0.559 ± 0.076c | 19.10 ± 3.47b |
| C-2h | 3104.01 ± 1175.45b | 82.33 ± 6.05b | 375.40 ± 65.98bc | 0.562 ± 0.066c | 18.10 ± 3.43b |
| C-5h | 2458.88 ± 699.99bc | 68.00 ± 10.86c | 317.80 ± 71.98cd | 0.699 ± 0.065b | 18.30 ± 2.89b |
| C-8h | 2436.24 ± 943.16c | 70.06 ± 6.45c | 339.80 ± 61.09c | 0.695 ± 0.068b | 18.06 ± 1.68b |
| C-24h | 1940.78 ± 482.52c | 70.03 ± 7.65c | 236 .89 ± 75.53d | 0.756 ± 0.047a | 21.98 ± 1.81ab |
Cellulose, hemicellulose and lignin are the main constituents of plant fibre. To compare the effects of steam explosion on these major chemical constituents of betel nut under different conditions, cellulose content was determined using the nitric acid-ethanol method, hemicellulose by the hydrochloric acid-DNS reducing sugar method and lignin by the concentrated sulfuric acid method (Klason's method). The results are shown in Table 3. For hemicellulose, the standard curve for glucose was as follows: Y = 0.7901X + 0.0428 (where X is the glucose concentration (mg/mL), Y is the absorbance value, R2 = 0.9990, n = 8). The cellulose content significantly increased with increasing pressure holding time and explosion pressure. After A-110 s and B-0.95 MPa treatment, the cellulose content increased from 27.12–29.87% and 30.49%, respectively. Unfortunately, the differences were not significant when comparing the soaked samples to the untreated samples. The lignin and hemicellulose contents decreased significantly with increasing pressure holding time and explosion pressure, and were negatively correlated with the initial moisture content. After the A-110s treatment, hemicellulose and lignin decreased from 45.43% and 25.89–41.38% and 23.38%, respectively, but those of other substances increased. This finding is consistent with that of Sun et al.(2015). Under steam explosion at high temperature, high pressure and high humidity, studies have shown that hemicellulose, a heterogeneous multimer consisting of many different monosaccharides, is the most unstable and prone to thermal hydrolysis, producing xylose, arabinose, glucose, galactose, furfural or hydroxymethylfurfural, etc., and hydrolysis of acetyl groups generates organic acids, which also accelerates the decomposition of other substances(Jacquet et al., 2015; Sun et al., 2015). As phenolic polymers, lignin undergoes a series of cleavages, rearrangements and breakdowns that can produce phenolics or phenolic acids and benzene derivatives(Dong et al., 2020). Additionally, cellulose undergoes thermal degradation in which the glycosidic bonds are cleaved to form monosaccharides, and the breaking of the C-C bond in furanose forms small molecules, as similarly occurs in hemicellulose, thereby reducing the absolute cellulose content. However, hemicellulose and lignin degraded more than cellulose, such that the relative content of cellulose increased after the steam explosion. These effects were closely related to the intensity of the steam explosion(Jacquet et al., 2011).
| Samples | Cellulose (%) | Hemicellulose (%) | Lignin (%) |
|---|---|---|---|
| BN | 27.12 ± 0.55c | 45.43 ± 1.11a | 25.89 ± 0.96a |
| A-0s | 27.90 ± 0.86bc | 44.52 ± 1.31ab | 24.96 ± 0.55ab |
| A-5s | 27.55 ± 0.84bc | 43.00 ± 0.91abc | 24.55 ± 0.47abc |
| A-20s | 28.84 ± 0.69ab | 42.90 ± 0.64bc | 24.16 ± 0.80bc |
| A-50s | 28.37 ± 0.55abc | 43.05 ± 0.93abc | 24.18 ± 0.81bc |
| A-80s | 29.78 ± 1.01a | 41.51 ± 0.86c | 24.34 ± 0.70bc |
| A-110s | 29.87 ± 0.98a | 41.38 ± 1.18c | 23.38 ± 0.61c |
| BN | 27.12 ± 0.55c | 45.43 ± 1.11a | 25.89 ± 0.96a |
| B-0.15MPa | 27.88 ± 0.91c | 45.14 ± 1.18ab | 25.02 ± 1.16ab |
| B-0.25MPa | 28.71 ± 0.51c | 43.28 ± 1.17ab | 24.05 ± 0.85bc |
| B-0.35MPa | 28.37 ± 0.55bc | 43.05 ± 0.93ab | 24.18 ± 0.81bc |
| B-0.55MPa | 27.92 ± 0.97c | 42.73 ± 1.20b | 23.62 ± 0.46bc |
| B-0.75MPa | 29.88 ± 1.10ab | 43.87 ± 0.78ab | 24.00 ± 0.99bc |
| B-0.95MPa | 30.49 ± 1.33a | 42.61 ± 0.37b | 22.97 ± 0.90c |
| BN | 27.12 ± 0.55a | 45.43 ± 1.11a | 25.89 ± 0.96a |
| C-0h | 28.37 ± 0.55a | 43.05 ± 0.93b | 24.18 ± 0.81a |
| C-2h | 28.60 ± 0.52a | 44.03 ± 0.48ab | 24.59 ± 1.07a |
| C-5h | 27.83 ± 0.80a | 44.18 ± 1.37ab | 24.29 ± 1.03a |
| C-8h | 27.09 ± 1.28a | 45.17 ± 0.76a | 25.10 ± 0.72a |
| C-24h | 27.31 ± 1.08a | 44.54 ± 0.97ab | 25.92 ± 0.90a |
Several reports have shown that polyphenols, as important active substances, are widely present in plant flowers, fruits, leaves, and bark and can exert antioxidant activity by inactivating lipid radicals or preventing the decomposition of hydroperoxides(Pitchaon et al., 2007). The results of the Folin-Phenol method for the free and bound phenols of betel nut are shown in Fig. 5. The gallic acid standard curve is Y = 0.0067X + 0.0054 (where X is the concentration of gallic acid (µg/mL), Y is the absorbance value R2 = 0.9991, n = 5). As seen from the results, the free phenol content decreased significantly with increasing pressure holding time and explosion pressure; interestingly, the free phenol content increased and then decreased with increasing initial moisture content compared to the unsoaked sample. The largest decrease was observed after the A-110s treatment, from 34.32 mg (GAE)/g of the untreated sample to 16.77 mg (GAE)/g, a decrease of more than half. The greatest increase in free phenol content, compared to the unsoaked samples, was observed after D-5 h treatment, from 21.58 mg (GAE)/g to 29.79 mg (GAE)/g. However, the free phenol content was still significantly lower than that of the untreated samples. The bound phenol content in betel nut was only 22.12% of the total phenol (free phenol + bound phenol) of 44.07 mg (GAE)/g, which was only 9.75 mg (GAE)/g. The greatest decrease in total phenol content was observed with the A-110s treatment at only 25.68 mg (GAE)/g. These results corroborate the findings of previous work in heat treatment that resulted in a reduction in the phenolic content. This may be attributed to the fact that phenolic compounds are prone to degradation or polymerization in steam explosion treatment, as determined by the aromatic ring type and orientation(Kim & Mai, 2020). However, it is surprising that this result differs somewhat from Gong et al.(2012), who assumed that the phenol content increased under certain conditions due to the steam explosion treatment inactivating some oxidases and breaking some bonds, facilitating phenolic release.
As the nitrogenous alkaline organic compounds present in plants, alkaloids in betel nut mainly include four alkaloids (Arecoline, Arecaine, Guvacoline, Guvacine)(Chen et al., 2021). The chromatograms of the betel nut and the highest concentration of the mixed standards are shown in Fig. 6. According to the results of the determination of betel nut major alkaloids in Table 4 and Fig. 7, it is noteworthy that all four alkaloids decreased significantly with increasing initial moisture content, and D-24h treatment the total alkaloid content decreased from 7.84 mg/g to 5.81 mg/g. This could be attributed to the fact that alkaloids usually have a higher lipid solubility, but the water solubility not being negligible. A similar finding was also reported by Carvajal-Larenas et al.(2014). Arecoline was relatively stable and Guvacoline only showed a significant decrease during high-pressure treatment, while Arecaine and Guvacine showed a significant decrease with increasing pressure holding time and explosion pressure. After A-110s treatment, the contents of Arecaine and Guvacine decreased by 19.31% and 45.09%, respectively. This finding is consistent with that of Shetge et al.(2020), who compared several heat-treated Poppy seeds, which showed different degrees of decrease in alkaloids, and speculated that one of the reasons for this was the promotion of oxidation leading to thermal degradation.
| Samples | Arecoline (mg/g) | Arecaine (mg/g) | Guvacoline (mg/g) | Guvacine (mg/g) |
|---|---|---|---|---|
| BN | 3.65 ± 0.21a | 1.45 ± 0.10a | 0.50 ± 0.02a | 2.24 ± 0.02a |
| A-0s | 3.67 ± 0.19a | 1.21 ± 0.12b | 0.45 ± 0.04ab | 1.65 ± 0.10b |
| A-5s | 3.58 ± 0.18a | 1.27 ± 0.13ab | 0.43 ± 0.02b | 1.62 ± 0.05b |
| A-20s | 3.71 ± 0.23a | 1.24 ± 0.02b | 0.51 ± 0.04a | 1.60 ± 0.05b |
| A-50s | 3.56 ± 0.15a | 1.12 ± 0.03b | 0.50 ± 0.05a | 1.32 ± 0.01cd |
| A-80s | 3.74 ± 0.19a | 1.21 ± 0.08b | 0.48 ± 0.04ab | 1.36 ± 0.03c |
| A-110s | 3.53 ± 0.08a | 1.17 ± 0.07b | 0.46 ± 0.03ab | 1.23 ± 0.03d |
| BN | 3.65 ± 0.21ab | 1.45 ± 0.10ab | 0.50 ± 0.02ab | 2.24 ± 0.02a |
| B-0.15MPa | 3.75 ± 0.13a | 1.48 ± 0.07a | 0.52 ± 0.01a | 2.18 ± 0.05b |
| B-0.25MPa | 3.73 ± 0.09a | 1.31 ± 0.11bc | 0.54 ± 0.04a | 2.22 ± 0.02ab |
| B-0.35MPa | 3.56 ± 0.15ab | 1.12 ± 0.03d | 0.50 ± 0.05ab | 1.32 ± 0.01c |
| B-0.55MPa | 3.57 ± 0.10ab | 1.22 ± 0.09cd | 0.51 ± 0.04a | 1.21 ± 0.04d |
| B-0.75MPa | 3.67 ± 0.13a | 1.37 ± 0.04cd | 0.53 ± 0.02a | 1.31 ± 0.02c |
| B-0.95MPa | 3.41 ± 0.12b | 1.13 ± 0.03d | 0.45 ± 0.02b | 1.30 ± 0.01c |
| BN | 3.65 ± 0.21a | 1.45 ± 0.10a | 0.50 ± 0.02a | 2.24 ± 0.02a |
| C-0h | 3.56 ± 0.15ab | 1.12 ± 0.03c | 0.50 ± 0.05a | 1.32 ± 0.01d |
| C-2h | 3.70 ± 0.04a | 1.26 ± 0.04b | 0.53 ± 0.03a | 1.45 ± 0.02c |
| C-5h | 3.34 ± 0.09bc | 0.96 ± 0.03d | 0.41 ± 0.03b | 1.65 ± 0.03b |
| C-8h | 3.26 ± 0.08c | 1.13 ± 0.07c | 0.39 ± 0.02b | 1.50 ± 0.03c |
| C-24h | 3.00 ± 0.11d | 0.95 ± 0.04d | 0.39 ± 0.05b | 1.48 ± 0.04c |
The texture analyser is an instrument for measuring various mechanical properties of foods based on simulated chewing and is widely used in the analysis of food texture; however, there are few reports of using a texture analyser to simulate chewing. To explore the effect of steam explosion on the release behaviour of betel nut, A-50 s was selected as a representative sample after the steam explosion. Comparisons were made with untreated samples under the same release conditions to compare the differences in released substances. As the betel nut is hard and easily exceeds the maximum force range of the texture analyser forces, rendering testing incompatible, the betel nut was first soaked and then the analyser experiment was conducted. And there was no significant difference in the Chemical composition of the solutions after sample soaking. To better simulate chewing, the number of experimental compressions and saliva additions was determined based on the observation of betel nut chewing behaviour and related literature(Chen, 2007).
In general, as shown in Fig. 8, the greatest quantity of substance was released between the 120th and 240th compressions in this simulated chewing condition. One possible explanation for this is that betel nut fibres were not yet loose and experienced less contact with the artificial saliva in the first 120 compressions, releasing a limited amount of substance; subsequently, simulated chews released progressively less material due to a decrease in the total amount of remaining substance. Comparing the release of betel nut before and after steam explosion treatment, the steam explosion promoted the release of soluble solids, total phenols, and total alkaloids, particularly when simulating chewing during the first 240 cycles. Soluble solids, total phenols and total alkaloids increased by up to 56%, 79% and 46%, respectively. There are several explanations for this result: steam explosion treatment disrupts the initially compact structure of betel nut, increases the contact area with artificial saliva, and facilitates material exchange(Adekunle et al., 2017); microstructure disruption also promotes the release of material. Adekunle et al.(2017) similarly found that steam explosion treatment of maize stems, enhanced the release of phenols. In addition, previous studies have shown that steam explosion promotes the release of dietary fibres and lipids, and other substances(Hu et al., 2020).
This study explored the effect of different pressure holding times, explosion pressures, and initial moisture contents on the softening effect of betel nut and the physical properties, microscopic morphology, and chemical compositions. We showed that the explosion efficiency positively correlated with the pressure holding time and explosion pressure, while the initial moisture content reduced the explosion effectiveness. The research also showed that the fibre hardness and Shore hardness decreased by 56.17%-89.28% and 7.03%-34.29%, respectively, and the transverse tensile strength and fibre tensile strength also decreased, while the coefficient of friction and the moisture content increased. After steam explosion, the betel nut increased in transverse diameter, became darker and more yellow‒red in colour, and showed a damaged microstructure. Cracks and cavities appeared in the epidermis and inter-fibre of the betel nut. Lignin, hemicellulose, free phenols, bound phenols and alkaloids content decreased after steam explosion treatment. Under strong steam explosions, the cellulose content increased slightly. The steam explosion increased the amount of phenols, alkaloids and soluble solids released in betel nut under the same simulated release conditions. These results suggested that steam explosion was very effective in softening the betel nut, but in practice, excessive steam explosion yielded more crumbs when the betel nut was chewed. This study provided a comprehensive evaluation of steam explosion technology for betel nut softening, which will help to better apply steam explosion in this field. Steam explosion technology was used for the purpose of softening fibres, an application which can expand the use of steam explosion. Further experiments need to be designed to determine the optimum conditions for steam explosion to soften betel nut fibres, based on which other methods of fibre softening can be explored. In general, steam explosion to soften betel nut is an effective and feasible method that can help to reduce the hazards due to the excessive hardness of the fibre of betel nut and contribute to the green and healthy development of the Chinese betel nut industry.
Acknowledgments
The authors thank the people of the Fruit and Vegetable Processing Team at the School of Food Science and Engineering of Hainan University and Hainan Huachuang Areca Nut Research Institute.
Funding Declaration
This work was supported by National Natural Science Foundation of China (32260598) and Hainan Province Science and Technology Special Fund (ZDYF2022XDNY166).
CRediT authorship contribution statement
Bowen Yang: Data curation, Formal analysis, Writing - original draft. Yaping Xu: Data curation, Methodology, Visualization. Weijun Chen: Funding acquisition, Resources, Supervision. Wenxue Chen: Project administration, Supervision, Validation. Qiuping Zhong: Resources. Supervision, Validation. Ming Zhang: Resources, Supervision. Jianfei Pei: Supervision. Haiming Chen: Conceptualization, Funding acquisition, Methodology, Project administration, Writing - review & editing.
Conflicts of Interest
No conflicts of interest are declared for any of the authors.
Data Availability Statement
The data presented in this study are available on request from the corresponding author.