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Effect of high-pressure homogenization on the extraction of sulforaphane from broccoli (Brassica oleracea) seeds
PTEC-13955; No of Pages 7 Powder Technology xxx (2018) xxx
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Effect of high-pressure homogenization on the extraction of sulforaphane from broccoli (Brassica oleracea) seeds Jun-jie Xing a,b, Yan-ling Cheng c,d, Paul Chen c, Lei Shan e, Roger Ruan c, Dong Li a,⁎, Li-jun Wang f,⁎ a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Engineering, National Energy R & D Center for Non-food Biomass, China Agricultural University, P. O. Box 50, 17 Qinghua Donglu, Beijing 100083, China School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China c Center for Biorefining and Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN 55108, USA d Biochemical Engineering College, Beijing Union University, Beijing, China e Department of Food science and Nutrition, University of Minnesota, St. Paul, MN 55108, USA f College of Food Science and Nutritional Engineering, Beijing Key Laboratory of Functional Food from Plant Resources, China Agricultural University, Beijing, China b
a r t i c l e
i n f o
Article history: Received 12 March 2018 Received in revised form 2 December 2018 Accepted 3 December 2018 Available online xxxx Keywords: High-pressure homogenization Broccoli seeds Sulforaphane extraction High-speed shear.
a b s t r a c t High-pressure homogenization (HPH) has the potential to improve the exaction yield of bioactive compound from food and food waste. This study investigated the use of HPH or microfluidization as an alternative assisted method for sulforaphane extraction from raw broccoli seeds. The mean particle size, morphological characteristics, and extraction yields of all samples processed at different HPH pressure levels (3000-23,000 psi) and passes (1–5) were examined. After HPH, the particle size of broccoli seeds was reduced 2–10 times and the particle size distribution pattern also changed from bimodal to unimodal with increasing pressures and number of passes. The highest sulforaphane content obtained with HPH at 5000 psi and 5 passes was 2199 μg sulforaphane/g broccoli seeds, which is 3 times more than the control one. Particular interest was given to the relationship between sulforaphane yield and particle size. Generally, the extraction yield increased with decreasing particle size; however, excessive size reduction did not necessarily result in significant increase in sulforaphane content. In addition, the pressure is not the higher the better (b8000 psi, 55 MPa). Scanning electron morphology (SEM) also confirmed that cell rupture and cell walls breakage occurred during the HPH process. © 2018 Elsevier B.V. All rights reserved.
1. Introduction In the past decades, sulforaphane (SF) has attracted substantial attention thanks to its antioxidant, anti-inflammatory, and anticarcinogenic properties [1]. SF is an isothiocyanate derived from glucoraphanin (GR) and has been proved to be one of the best natural inducer of phase II enzymes in human; these enzymes can detoxify cancer-causing chemicals thus protect against carcinogens and inflammation [2]. When consumed, the plant tissues and cells are damaged and GR comes into contact with one endogenous enzyme, myrosinase (thioglucoside glucohydrolase, EC 3.2.1.147) [3]; then GR is hydrolyzed directly into SF under certain conditions. GR richly exists in Brassica oleracea vegetables, such as broccoli, cabbage, cauliflower [4], among which, seeds from broccoli cultivars are the most likely source for GR as compared with other parts of cruciferous vegetables [1,5,6]. Many studies have aimed at improving the extraction efficiency of sulforaphane from different plant resources. On the one hand, there is no doubt that the hydrolysis reaction conditions like pH, ⁎ Corresponding authors. E-mail addresses: [email protected] (D. Li), [email protected] (L. Wang).
epithiospecifier protein (ESP) activity, Fe2+, ascorbic acid, time, and temperature can influence conversion efficiency of GR to SF [1,4,7]. On the other hand, since it requires a longer time for the conventional extraction method, some assisted extraction techniques with higher extraction efficiency should be explored to enhance the yield and bioactivity of desired products [8]. Recently, several assisted methods have been employed to improve the extraction efficiency of bioactive compounds from plant seeds, including gamma irradiation, ultrasonication, microwave, hydrostatic pressure, and dynamic pressure homogenization [8–11]. Teh and Birch [12] found that ultrasonic treatment (UA) at room temperature increased the extraction content of polyphenol from hemp, flax and canola seeds cake. Chemat et al. [13] used a microwave-assisted method (MW) to extract the carvone and limonene from caraway seeds, and the cell destruction of seeds was observed from the scanning electron micrographs. Prasad et al. [14] also found that ultrasonication and high-pressure extractions had a potential to achieve high extraction efficiency from plant tissues. High hydrostatic pressure technique (HHP), with pressure ranging from 100 to 1000 MPa, has been widely used to enhance mass transfer [15,16] through increasing cell permeability as well as secondary metabolite diffusion [8]. Briones-Labarca et al. [8] used both ultrasonic extraction
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and high hydrostatic pressure methods to improve the release of bioactive compounds from papaya seeds. Extraction of antioxidant and anticancer compounds from plant seeds is a challenge because of the presence of rigid shell of the seeds and the difficulty in reducing the particle size [17,18]. Compared with traditional extraction techniques, the above methods each has their own advantages like time-saving, high efficiency, and high extraction yields. The UV, MW, and HPP methods could enhance the mass transfer and release the bioactive compounds from plant tissues, either by cavitation force [12], rapid rise of temperature in plant cells [19], or high hydrostatic pressure [15], respectively. Moreover, one dynamic high pressure technique has been developed to assist in extracting intracellular components from food and food waste [20,21]. Unlike hydrostatic pressure, high-pressure homogenization (HPH, 20-200 MPa) or microfluidization forces fluid to continuously flow through a homogenizing valve using a positive displacement pump [18,22]. HPH has been reported to cause particle size reduction and was used for pharmaceuticals, food, and other materials, manufacturing industries [23]. HPH can disrupt cellular structures of plant seeds and help release the bioactive compounds into the extracellular environment. However, reports focusing on the application of high-pressure homogenization in sulforaphane extraction are limited. During HPH process, high velocity micro-streams can result in high shear stress when passing through the narrow channel of the chamber [24]. The subsequent resultant cavitation, shear, turbulence and shock waves could lead to the disruption of suspended particles and the release of cellular products [20,22,25]. Furthermore, the ultra-high pressure up to 200 MPa may also impose the similar HHP effect on the seeds tissue, thus increasing the access of solvent to cellular materials [21]. Therefore, the high-pressure homogenization has the potential of improving the extraction efficiency of sulforaphane from plant seeds. The present work was to study the feasibility of using high-pressure homogenization as an assisted method to increase the extraction yield of sulforaphane from broccoli seeds. The particle size and distribution of broccoli seeds after high-pressure homogenization process with different pressures and passes were analyzed. The changes in the morphological structure were also studied using scanning electron micrographs (SEM). Particular interest was given to the relationship between the sulforaphane yield and the particle size. 2. Materials and methods 2.1. Materials and chemicals Fresh Brassica oleracea (SBC2-4AK) seeds were acquired from Todds Seeds (Novi, MI). Following chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO): sulforaphane standard at ≥98% purity, HPLC-grade acetonitrile (ACN), methanol, deionized water, analytical grade (AG) ethanol, AG ethyl acetate, AG hexane, AG anhydrous sodium sulfate, and phosphate buffered saline (PBS). 2.2. Extraction methods 2.2.1. High speed shearing pretreatment (HSS) High speed shearing was used in advance prior to HPH. In this procedure, 10 mM phosphate buffered saline (PBS) solution was prepared by dissolving 1 pack PBS powder in 5 l of distilled water at 25 °C. Broccoli seeds (4 g) were immersed into the PBS (200 mL) solutions at room temperature (pH = 7.2) for 30 min to soften the hard shell of seeds. Seeds were homogenized with a high speed shear mixer (T25 Ultraturrax, IKA Works, Inc. USA) at 11000 rpm for 30 s. This HSS procedure was followed by the high-pressure homogenization procedure described below. 2.2.2. High-pressure homogenization (HPH) HSS treated samples were then transferred to a high-pressure microfluidizer (M110-Y, Microfluidics Corp. USA) equipped with a Z
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type chamber (H10Z, 100 μm), and homogenized with the different pressures of 3000, 5000, 8000, 10,000, 13,000, 18,000, and 23,000 psi (20-160 MPa) and passes of 1, 2, 3, 4, and 5, respectively. In this study, HPH Samples were abbreviated and named as “3000-1”, which means the samples were treated under the pressure of 3000 psi and 1 pass, etc. 2.2.3. Seeds extraction In this work, broccoli seed treated by high speed shearing (HSS) but without HPH was defined as the control sample. The control and HPH samples were allowed to autolyze for 30 min or 8 h at 25 °C. After autolyzing, the suspension was first vacuum-filtered, and then the wet cake of broccoli seeds was washed and extracted two times with 200 mL of ethyl acetate, which was combined with the former filtrate. The ethyl acetate layers were separated and dried at 35 °C under vacuum in a rotary evaporator Büchi R II (Büchi, Switzerland). The residue was dissolved in 100 mL 10% ethanol in water (v/v) and washed two times with equal volumes of hexane to remove nonpolar contaminants. The ethyl acetate layers were pooled, dried over anhydrous sodium, and dried at 35 °C under vacuum in a rotary evaporator. The residue was dissolved in 25 mL methanol solvent and was then filtered through a 0.45 μm membrane filter prior to injection into HPLC. 2.3. Analysis methods 2.3.1. High performance liquid chromatography (HPLC) Sulforaphane was analyzed with an Agilent Infinity 1260 HPLC (USA), equipped with an Agilent 1260 Quat pump and an Acclaim C30 column (Thermo Fisher Scientific Inc. Waltham, MA), 150 mm × 4.6 mm, 3 μm. The solvent system consists of 20% acetonitrile in water, and then changes linearly over 10 min to 60% acetonitrile, and maintained 100% acetonitrile for 2 min to purge the column. Column oven temperature was set at 30 °C. The flow rate was 1 mL/min, and 10 μL portions were injected into the column. Sulforaphane was detected by UV 254 nm. 2.3.2. Standard curve preparation of sulforaphane A stock solution was prepared with 5 mg of sulforaphane reference standard, which was dissolved and diluted in 5 mL acetonitrile. The desired volumes of the standard stock solution of sulforaphane were pipetted into 1.5 mL Eppendorf Tubes and diluted with 1 mL acetonitrile. The final concentrations of sulforaphane were in the range of 15.625–1000 μg/mL. A 10 μL portion of each solution was subjected to HPLC-UV in 6 replicates, and a cali1bration curve was made. Each solution was injected in duplicate. Peak areas were recorded for all the solutions. Quantification was carried out on the basis of the external standard method. 2.4. Particle size measurements The mean particle sizes of the HPH treated broccoli seeds with different pressures and passes were determined using an LS-13-320 laser diffraction particle size analyzer (Backman Coulter Inc., Brea, CA) with the universal liquid module. The mean of the particle size was calculated from the triplicates. The particle size distribution was also recorded. 2.5. Scanning electron microscopy The morphology of broccoli seeds treated with high-pressure homogenization after vacuum drying was investigated using scanning electron microscope (SEM, JSM-6500F, JEOL). Samples were mounted with double-sided carbon adhesive tabs on aluminum stubs, and sputter coated with gold‑palladium (60%-40%). The sample patterns were observed at an accelerating voltage of 2 or 5 kV.
Please cite this article as: J. Xing, Y. Cheng, P. Chen, et al., Effect of high-pressure homogenization on the extraction of sulforaphane from broccoli (Brassica ole..., Powder Technol., https://doi.org/10.1016/j.powtec.2018.12.010
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2.6. Statistical analysis All experiments were carried out in triplicate and the results were expressed as mean ± standard deviation. SPSS 17.0 software (SPSS Inc., Chicago, USA) was used for analysis of variance (ANOVA) and significant differences were analyzed using Duncan's multiple range tests. A 0.95 confidence level (p b 0.05) was used to determine if a significant difference existed between two mean values.
particle size; and there was no difference among the particle size of samples 13,000 psi with different 3, 4, and 5 passes. Few intact cells would be left in the suspension of broccolis seeds when the actual particle size was reduced to around and smaller than the average cell size, which can be observed from the following morphological analysis. The disruption of big particles can result in the increase in specific surface area [11], which means more content of components (GRs) and endogenous enzymes will be released from the cells and compartments.
3. Results and discussion
3.2. Particle size distribution (PSD)
3.1. Particle size reduction
The particle size distribution (PSD) of some representative broccoli seeds samples were represented in Fig. 1. The broccoli seeds of control sample presented a bimodal size distribution profile ranging from 1 to 1000 μm. Broccoli seeds contain two components: the outside shell and the inside kernel. Based on the observation of wet cake of samples after the vacuum filtration (Fig. 2), the first peak of control sample was supposed to represent the size of broccoli kernel (around 10 μm) while the second one referred to the broccoli shells (around 400 μm). During the homogenization process, the broad distribution profile of particle size gradually became narrower. With increasing processing pressure and pass, the bimodal distribution model of particle size was finally converted to single peaked, thus producing the desired results, including uniform particle and size reduction. In order to explain this observation, a series of images of samples before and after homogenization with various conditions were analyzed. The wet cake images of broccoli seeds are shown in Fig. 2. With the increase of homogenization pressure, a more uniform particle size of HPH samples could be observed; the bicolor of the wet cake was gradually changing to monotonous one. Therefore, we can speculate that the particle size reduction at the beginning of homogenization was mainly due to the tissues crush of shell parts. During the homogenization process, not only the high shear force in the small chamber channel can tear up the tissue rupture [18], but the dynamic pressure on the cellular tissue also impose the similar effect with hydrostatic pressure on the seeds tissue, which may increase the mass transfer of the solvent into materials and the soluble constituents (GR and myrosinase) into solvent [15].
A preliminary study was aimed to detect whether homogenization process had an effect on the size reduction of broccoli seed since processing broccoli seeds with homogenizer is a great challenge because of the presence of rigid shell of the seeds [14,18]. Before this homogenization experiment, the broccoli seeds were immersed in the PBS solution to soften the broccoli seeds. Subsequently, the high speed shearing was used to reduce the particle size of broccoli seeds in advance before going through the homogenizer. It was also reported that the temperature increase during homogenization also helped soften the cell walls and then facilitate the cell disruption [18]. The particle sizes of the broccoli seeds treated with high-pressure homogenization under different pressures (3000–23,000 psi) and passes (1–5 passes) were listed in Table 1. HSS process could effectively break the whole broccoli seeds to small particles of 357 μm. In general, the mean size of broccoli seeds was greatly reduced after HPH pretreatment. The results also showed that HPH combined with high speed shearing pre-treatment achieved the desired level of particle size reduction. Compared with the control sample, the particle size of sample 3000-1 (homogenization with 3000 psi and 1 pass) was reduced by 2 times to 170 μm. Microfluidizer processor is a lab machine that provides high shear rates to maximize the energy-per-unit fluid volume to produce uniform micron particles [26,27]. When employed for cell disruption, the high-pressure homogenization process involves mainly two key factors: pressure and pass [18,22,25]. Table 1 showed that pressure could exert more significant influence on the particle size reduction than the pass did. Even though the particle sizes of samples 3000 psi were gradually reduced from 170 to 111 μm with the increase in pass from 1 to 5, the particle size of control sample can be directly reduced to 115 μm at 5000 psi for only one pass. In the meantime, the size of sample 5000-4 is almost the same with sample 8000-1, and 8000-2 with 13,000-1. On the other hand, the influence of pressure on the particle size declined with increasing number of passes, especially at higher pressure conditions. For example, there is no difference among the particle sizes of sample 13,000-2, 18,000-2, and 23,000-2. Similarly, the effect of the pass on the particle size also varied with the pressure powered by the pump. For all pressure levels, the particle size can be reduced to some extent when the process passes were increased from 1 to 2 or 3. Nevertheless, more passes did not necessarily result in smaller
3.3. Linearity of standard curve The calibration curve on standard sulforaphane solutions showed that there was good linearity over the sulforaphane concentration range from 15.625 to 1000 μg/mL (Fig. 3). And the linear regression analysis of the peak area response (y) versus the theoretical concentration (x) gave the following equation: y = 1.577 × −2.885, r2 = 0.9999. The system precision was analyzed by assaying 6 injections of standard solution and calculating the relative standard deviation (RSD) of the peak area response [28]. The RSD (%) for standard sulforaphane was 1.98. The sulforaphane peak in each broccoli seed extract chromatogram was identified based on the retention time of the standard solution. The similarity of chromatograms between the standard and
Table 1 Particle size (Mean size) of broccoli seeds treated by high-pressure homogenization with different pressure and passes*. Z-Avg (d μm) Pressure (psi)
Pass 1
Pass 2
Pass 3
Pass 4
Pass 5
Control 3000 5000 8000 10,000 13,000 18,000 23,000
357.0 ± 1.9a 170.6 ± 1.7b, A 115.3 ± 1.4c, A 82.8 ± 0.3d, A 76.6 ± 1.0e, A 64.3 ± 0.4f, A 60.7 ± 0.9g, A 52.1 ± 0.5h, A
125.4 ± 1.2a, B 85.1 ± 1.7b, B 64.3 ± 0.9c, B 58.2 ± 0.6d, B 44.0 ± 0.4e, B 43.6 ± 1.9e, B 44.1 ± 1.2e, B
122.5 ± 1.0a, B 61.1 ± 0.6b, C 52.0 ± 0.9c, C 48.3 ± 0.6d, C 36.3 ± 0.6e, C 38.2 ± 0.5e, C 37.1 ± 1.3e, C
112.4 ± 2.0a, C 60.0 ± 1.5b, CD 45.0 ± 0.8c, D 43.2 ± 0.8c, D 36.1 ± 0.5d, C 35.0 ± 0.7d, D 31.1 ± 1.7e, D
111 ± 0.2a, C 57 ± 1.4b, D 42 ± 1.5c, E 44 ± 0.7c, D 37 ± 1.7d, C 33.6 ± 1.3d, D 29 ± 1.9e, D
*, Different lowercase and capital letters within the same column differ significantly (p b 0.05). Values represent mean ± S.D., n = 3.
Please cite this article as: J. Xing, Y. Cheng, P. Chen, et al., Effect of high-pressure homogenization on the extraction of sulforaphane from broccoli (Brassica ole..., Powder Technol., https://doi.org/10.1016/j.powtec.2018.12.010
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Fig. 1. Particle size distribution of high-pressure homogenization treated broccoli seeds. Samples 3000-1 means the broccoli seeds were treated by HPH at 3000 psi and 1 pass, etc.
sample was used to evaluate the suitability of this HPLC method. The HPLC chromatograms of the sulforaphane standards (A) of the theoretical concentration of 500 μg/mL and broccoli seeds extracts of control sample (B) and 5000-5 (C) are shown in Fig. 4. Both the standard and sample presented a single peak at 5.1 min, with no interfering peaks on either side of the target peak. These results indicate that the HPLC method can be used for the quantitative analysis of sulforaphane in broccoli seeds, which is similar to the findings confirmed by Liang et al. [29]. 3.4. Extraction yield (EY) The sulforaphane extracted with ethyl acetate for each sample was quantitated by HPLC method. The HPLC peak area of sulforaphane extract from different broccoli seed samples were recorded and analyzed. The extraction yield was then calculated and expressed as the content (μg) of sulforaphane/weight (g) of raw broccoli seeds. The fresh weight (FW) was used because it gives a better idea of the content really ingested. Table 2 listed the sulforaphane contents in broccoli seeds with homogenization extraction method.
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Fig. 3. The standard curve obtained from sulforaphane analysis by HPLC.
In this study, the effect of high-pressure homogenization with different pressures and passes on the sulforaphane extraction was investigated. The results demonstrated that HPH could be used as an assisted method to help extract sulforaphane from broccoli seeds. Table 2 showed that the extraction yield was greatly increased after high-pressure homogenization. After gentle homogenization, the sulforaphane content of 3000-1 has more than doubled from 549 to 1481 μg/g than the control one. The highest content (2199 μg/g) of sulforaphane was found in sample 5000-5, which was three-fold more than the control one without HPH process. Furthermore, the pressure and passes applied also affected the final extraction yield. Regardless of passes, the extraction yield increase with increasing pressure levels and the mean sulforaphane content of samples under 3000, 5000, and 8000 psi were 1606, 1900, and 1966, respectively. However, the similarity with the effect of pressure on the particle size, higher pressure (13,000, 18,000, 23,000 psi) did not result in a significant increase in yield. At the meantime, the extraction yield can be always increased by increasing the process pass. For instance, the sulforaphane content of sample 3000-5, 5000-5, and 8000-5 were increased by 15.6%, 26.2%, and 13.7% compared with the sample 3000-1, 5000-1, and 8000-1, respectively.
Fig. 2. Wet cake images of HPH broccoli seeds after vacuum filtration process. A-F, Control, 3000-1, 5000-1, 8000-1, 13000-1, 23000-1. Samples 3000-1 means the broccoli seeds were treated by HPH at 3000 psi and 1 pass, etc.
Please cite this article as: J. Xing, Y. Cheng, P. Chen, et al., Effect of high-pressure homogenization on the extraction of sulforaphane from broccoli (Brassica ole..., Powder Technol., https://doi.org/10.1016/j.powtec.2018.12.010
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Fig. 4. The HPLC Chromatograms of the sulforaphane standard (A) of theoretical concentration of 500 μg/mL, broccoli seeds extract of Control (B), and sample 5000-5 (C). Conditions described in HPLC section.
Particular interest was paid to the relationship between the extraction yield and the particle size. The particle size changes are in agreement with the results of extraction yield at the lower pressure homogenization conditions (3000 psi). It is a really interesting finding that the particle size of sample 3000-1 was decreased two times and, at the same time, the sulforaphane extraction yield doubled the content of control sample. However, after this, the extraction yield did not increase at the same pattern with decreasing particle size. For example, the particle size of 23,000-5 has decreased up to 10 times (Table 1) while the highest extraction yield of sample 5000-5 was just 3 times more than the control one. This is because particle size could be reduced when seed tissues were broken down under the shearing and tearing effect, and cell disruption happened during this homogenization process [11]. Ideally, once the particle size of broccoli seeds was reduced to the point that it equals to the mean cell size, the target components contained in all the separated cells will be quite easy to extract. Beyond this point, any increase in pressure or pass will result in all the cell walls Table 2 Sulforaphane contents in broccoli seeds treated with or without high-pressure homogenization.⁎ Samples
Peak area
Peak time (min)
Yield (μg/g)
Control-30 min
135.7
5.04
549.2⁎
Control-8 h 3000–1 3000–3 3000–5 5000–1 5000–3 5000–5 8000–1 8000–3 8000–5 13,000–1 18,000–1 23,000–1
168.6 370.9 407.4 429.4 436.8 441.0 542.1 476.5 461.2 552.0 527.4 496.4 486.2
5.10 5.11 5.11 5.10 5.10 5.10 5.12 5.10 5.14 5.10 5.10 5.07 5.11
679.6 1481.4 1626.0 1713.2 1742.6 1759.2 2199.1 1899.9 1839.9 2159.9 1901.6 1978.8 1938.4
1606.9⁎⁎
1900.3
1966.5
⁎Two control samples treated with shearing process but without HPH were autolyzed for 30 min and 8 h, respectively. 3000–1, Sulforaphane extract of broccoli seeds treated at 3000 psi and 1 pass, etc. ⁎ Values represent the mean of three replicates per accession. ⁎⁎ Values represent the mean of same pressure level.
being broken and cells disrupted. It is easy to understand that all GRs and endogenous enzyme will be released to the reaction system and the highest yield of sulforaphane will become a matter of course under the selected optimum reaction conditions. Thus, extremely high pressures are helpful to reduce the particle size but not necessary to assist the sulforaphane extraction in terms of high-pressure homogenization. However, this does not prevent high-pressure homogenization from being a potential pretreatment of sulforaphane extraction. Here comes to another issue, what is the relationship between the mean cell size and the particle size of 5000-5 with highest sulforaphane yield? This is a key issue to the understanding of the extraction mechanism. To answer this question and to see what effect of dynamic pressure had on the cell walls and cells, the treated broccoli seeds were investigated under scanning electron microscope. 3.5. Morphology and structure We employed SEM to investigate the internal structural changes to broccoli seeds after the homogenization process. The effect of highpressure homogenization on the cell walls and cell destruction could be seen on SEM (500×) micrographs (Fig. 5). Before HPH, the broccoli seeds were broken to small particles (357 μm). The main part of this control sample seed tissues seemed remaining unaffected and most of the cells remained together without disruption. Smooth surface was also observed (Fig. 5A). After the homogenization process, broccoli cells were gradually damaged to different degrees; rough surface and cell disruption can be clearly observed in Fig. 5. Seeds tissues were disintegrated, disrupted, and randomly reassembled to form new smaller cell fragments. Furthermore, some large agglomerates with rough surface were observed. HPH even turned broccoli into flat thin chip-like structure (Fig. 5G); some gels-like cells are highly distorted and tightly aggregated (Fig. 5E, F). We noticed that there were no intact cells left in the system for sample 8000-5 (Fig. 5G), 13,000-1 (Fig. 5H), and 23,000-1 (Fig. 5I). This observation is similar to the report that the original complexes of polymers were totally broken into micro-fragments by homogenization, which also resulted in the irreversible disruption and aggregation of polymers [30]. There is no much difference between the samples treated with higher pressure (13,000 and 23,000 psi).
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Fig. 5. Scanning electron micrographs of broccoli seeds of control (A), 3000-1 (B), 3000-5 (C), 5000-1 (D), 5000-5 (E), 8000-1 (F), 8000-5 (G), 13000-1 (H), and 23000-1 (I) (10μm, 2 kV, 500× magnification).
Besides, we have scaled and measured the cell sizes from these SEM images by using the public software ImageJ 1.42q (National Institute of Health, USA) and found that the cell size of broccoli seeds was around 10-70 μm and the mean cell size was around 20-30 μm (data not shown). Considering both the sample 5000-5 (mean size, 57 μm) rather than other samples had the highest sulforaphane content (Table 2) and a few cells of sample 5000-5 (Fig. 5E) still remained their initial cell shape, we speculated that the particle size of samples do not have to be smaller than the mean cell size to favor the release of GR and endogenous enzymes. At 2000 and 5000× magnification, we also observed clear cell rupture (Fig. 6A), breakage at the edge of cell walls (Fig. 6B) and some
pores through the cell (Fig. 6C). High-pressure homogenization was reported using combined forces of shear, cavitation, and ultra-high pressures to process the samples [25]. These observations confirmed that the broccoli seeds underwent violent shearing and tearing during HSS and HPH [11]. Thus, the findings in this study suggested two extraction mechanisms of HPH: one is that homogenization reduced the particle size, increased the surface area of particles, and facilitated the contact between GR and endogenous enzymes; and the other involved the improvement of diffusion of the essential solvent (PBS solution) from the surface to the inside of the granular walls under the high pressure. All these influences could enhance the rate of mass transfer of cell constituents
Fig. 6. Scanning electron micrographs of broccoli seeds of sample 5000-3. Cell disruption, cell wall breakage, and pores were found in A, B, and C, respectively. (5 kV, 2000 and 5000× magnification).
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through the cell walls, which means more content of components and endogenous enzymes will be released from the cells. 4. Conclusions This study employed the high-pressure homogenization technique to assist in extraction of sulforaphane from broccoli seeds. High-pressure homogenization combined with high speed shear provided a better way of pretreating the broccoli seeds to smaller particle size than the conventional method. Varying degrees of size reduction were achieved by HPH with different pressures and passes. The analysis of the relationship between particle size and sulforaphane extraction yield shows that the sulforaphane content did not increase continuously with increasing pressure. In fact, pressure does not have to be very high (b8000 psi, 55 MPa) to successfully maximize the sulforaphane extraction. HPH also changed the original smooth surface to small flake-like structure and fragments. Cell rupture, cell walls breakage, and cell poration were observed in SEM images. The effect of HPH on the extraction was supposed duo to two mechanisms: cell disruption caused by high mechanical force and enhanced mass transfer rates across granular walls under high dynamic pressure. Both facilitated the contact between GR and myrosinase, which increase the sulforaphane yield from broccoli seeds. Thus in this study, high-pressure homogenization treatment showed potential application in extraction of sulforaphane from broccoli seeds. Acknowledgments This research was supported by National Natural Science Foundation of China (31771896). References [1] H. Liang, Q. Yuan, Natural sulforaphane as a functional chemopreventive agent: including a review of isolation, purification and analysis methods, Crit. Rev. Biotechnol. 32 (2012) 218–234. [2] H. Liang, C. Li, Q. Yuan, F. Vriesekoop, Application of high-speed countercurrent chromatography for the isolation of sulforaphane from broccoli seed meal, J. Agric. Food Chem. 56 (2008) 7746–7749. [3] J.W. Fahey, W.D. Holtzclaw, S.L. Wehage, K.L. Wade, K.K. Stephenson, P. Talalay, Sulforaphane bioavailability from glucoraphanin-rich broccoli: control by active endogenous myrosinase, PLoS One 10 (2015), e0140963. . [4] Z.-x. Gu, Q.-h. Guo, Y.-j. Gu, Factors influencing glucoraphanin and sulforaphane formation in brassica plants: a review, Integr. Agric. 11 (2012) 1804–1816. [5] H. Liang, Q. Yuan, Q. Xiao, Purification of sulforaphane from Brassica oleracea seed meal using low-pressure column chromatography, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 828 (2005) 91–96. [6] L. Zhansheng, L. Yumei, Z.Y. Fang, L. Mu Yang, M. Zrfg, Y.Y. Zhang, L.H. Honghao, Development and identification of anti-cancer component of sulforaphane in developmental stages of broccoli (Brassica oleracea var. italica L.), J. Food Nutr. Res. 4 (2016) 490–497. [7] C. Perez, H. Barrientos, J. Roman, A. Mahn, Optimization of a blanching step to maximize sulforaphane synthesis in broccoli florets, Food Chem. 145 (2014) 264–271. [8] V. Briones-Labarca, M. Plaza-Morales, C. Giovagnoli-Vicuña, F. Jamett, High hydrostatic pressure and ultrasound extractions of antioxidant compounds, sulforaphane and fatty acids from Chilean papaya (Vasconcellea pubescens) seeds: Effects of extraction conditions and methods, LWT Food Sci. Technol. 60 (2015) 525–534.
[9] V. Briones-Labarca, C. Muñoz, H. Maureira, Effect of high hydrostatic pressure on antioxidant capacity, mineral and starch bioaccessibility of a non conventional food: Prosopis chilensis seed, Food Res. Int. 44 (2011) 875–883. [10] R.H. Chen, J.R. Huang, M.L. Tsai, L.Z. Tseng, C.H. Hsu, Differences in degradation kinetics for sonolysis, microfluidization and shearing treatments of chitosan, Polym. Int. 60 (2011) 897–902. [11] X. Hua, S. Xu, M. Wang, Y. Chen, H. Yang, R. Yang, Effects of high-speed homogenization and high-pressure homogenization on structure of tomato residue fibers, Food Chem. 232 (2017) 443–449. [12] S.S. Teh, E.J. Birch, Effect of ultrasonic treatment on the polyphenol content and antioxidant capacity of extract from defatted hemp, flax and canola seed cakes, Ultrason. Sonochem. 21 (2014) 346–353. [13] S. Chemat, H. Aït-Amar, A. Lagha, D.C. Esveld, Microwave-assisted extraction kinetics of terpenes from caraway seeds, Chem. Eng. Process. Process Intensif. 44 (2005) 1320–1326. [14] K.N. Prasad, B.A.O. Yang, M. Zhao, N. Ruenroengklin, Y. Jiang, Application of ultrasonication or high-pressure extraction of flavonoids from litchi fruit pericarp, J. Food Process Eng. 32 (2009) 828–843. [15] H.-W. Huang, C.-P. Hsu, B.B. Yang, C.-Y. Wang, Advances in the extraction of natural ingredients by high pressure extraction technology, Trends Food Sci. Technol. 33 (2013) 54–62. [16] Z. Shouqin, Z. Junjie, W. Changzhen, Novel high pressure extraction technology, Int. J. Pharm. 278 (2004) 471–474. [17] K.N. Prasad, J. Hao, J. Shi, T. Liu, J. Li, X. Wei, S. Qiu, S. Xue, Y. Jiang, Antioxidant and anticancer activities of high pressure-assisted extract of longan (Dimocarpus longan Lour.) fruit pericarp, Innovative Food Sci. Emerg. Technol. 10 (2009) 413–419. [18] N. Samarasinghe, S. Fernando, R. Lacey, W.B. Faulkner, Algal cell rupture using high pressure homogenization as a prelude to oil extraction, Renew. Energy 48 (2012) 300–308. [19] W. Sookjitsumran, S. Devahastin, A.S. Mujumdar, N. Chiewchan, Comparative evaluation of microwave-assisted extraction and preheated solvent extraction of bioactive compounds from a plant material: a case study with cabbages, Innovative Food Sci. Emerg. Technol. 51 (2016) 2440–2449. [20] P. Comuzzo, S. Calligaris, L. Iacumin, F. Ginaldi, S. Voce, R. Zironi, Application of multi-pass high pressure homogenization under variable temperature regimes to induce autolysis of wine yeasts, Food Chem. 224 (2017) 105–113. [21] X. Huang, Z. Tu, Y. Jiang, H. Xiao, Q. Zhang, H. Wang, Dynamic high pressure microfluidization-assisted extraction and antioxidant activities of lentinan, Int. J. Biol. Macromol. 51 (2012) 926–932. [22] C.H. Karacam, S. Sahin, M.H. Oztop, Effect of high pressure homogenization (microfluidization) on the quality of Ottoman Strawberry (F. Ananassa) juice, LWT Food Sci. Technol. 64 (2015) 932–937. [23] X. Zhu, Y. Cheng, P. Chen, P. Peng, S. Liu, D. Li, R. Ruan, Effect of alkaline and highpressure homogenization on the extraction of phenolic acids from potato peels, Innovative Food Sci. Emerg. Technol. 37 (2016) 91–97. [24] B.C. Porto, P.E. Augusto, A. Terekhov, B.R. Hamaker, M. Cristianini, Effect of dynamic high pressure on technological properties of cashew tree gum (Anacardium occidentale L.), Carbohydr. Polym. 129 (2015) 187–193. [25] W. Liu, J. Liu, C. Liu, Y. Zhong, W. Liu, J. Wan, Activation and conformational changes of mushroom polyphenoloxidase by high pressure microfluidization treatment, Innovative Food Sci. Emerg. Technol. 10 (2009) 142–147. [26] C. Baldwin, C.W. Roblnsonf, Disruption of Saccharomyces cerevisiae using enzymatic lysis combined with high-pressure homogenizatio, Biotechnol. Tech. 4 (1990) 329–334. [27] A. Carlson, M. Signs, L. Liermann, R. Boor, K.J. Jem, Mechanical disruption of Escherichia coli for plasmid recovery, Biotechnol. Bioeng. 48 (1995) 303–315. [28] P. Kuang, D. Song, Q. Yuan, R. Yi, X. Lv, H. Liang, Separation and purification of sulforaphene from radish seeds using macroporous resin and preparative high-performance liquid chromatography, Food Chem. 136 (2013) 342–347. [29] H. Liang, C. Li, Q. Yuan, F. Vriesekoop, Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography, J. Agric. Food Chem. 55 (2007) 8047–8053. [30] J.L. Hu, S.P. Nie, M.Y. Xie, High pressure homogenization increases antioxidant capacity and short-chain fatty acid yield of polysaccharide from seeds of Plantago asiatica L, Food Chem. 138 (2013) 2338–2345.
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Effect of high-pressure homogenization on the extraction of sulforaphane from broccoli (Brassica oleracea) seeds Jun-jie Xing a,b, Yan-ling Cheng c,d, Paul Chen c, Lei Shan e, Roger Ruan c, Dong Li a,⁎, Li-jun Wang f,⁎ a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Engineering, National Energy R & D Center for Non-food Biomass, China Agricultural University, P. O. Box 50, 17 Qinghua Donglu, Beijing 100083, China School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China c Center for Biorefining and Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, MN 55108, USA d Biochemical Engineering College, Beijing Union University, Beijing, China e Department of Food science and Nutrition, University of Minnesota, St. Paul, MN 55108, USA f College of Food Science and Nutritional Engineering, Beijing Key Laboratory of Functional Food from Plant Resources, China Agricultural University, Beijing, China b
a r t i c l e
i n f o
Article history: Received 12 March 2018 Received in revised form 2 December 2018 Accepted 3 December 2018 Available online xxxx Keywords: High-pressure homogenization Broccoli seeds Sulforaphane extraction High-speed shear.
a b s t r a c t High-pressure homogenization (HPH) has the potential to improve the exaction yield of bioactive compound from food and food waste. This study investigated the use of HPH or microfluidization as an alternative assisted method for sulforaphane extraction from raw broccoli seeds. The mean particle size, morphological characteristics, and extraction yields of all samples processed at different HPH pressure levels (3000-23,000 psi) and passes (1–5) were examined. After HPH, the particle size of broccoli seeds was reduced 2–10 times and the particle size distribution pattern also changed from bimodal to unimodal with increasing pressures and number of passes. The highest sulforaphane content obtained with HPH at 5000 psi and 5 passes was 2199 μg sulforaphane/g broccoli seeds, which is 3 times more than the control one. Particular interest was given to the relationship between sulforaphane yield and particle size. Generally, the extraction yield increased with decreasing particle size; however, excessive size reduction did not necessarily result in significant increase in sulforaphane content. In addition, the pressure is not the higher the better (b8000 psi, 55 MPa). Scanning electron morphology (SEM) also confirmed that cell rupture and cell walls breakage occurred during the HPH process. © 2018 Elsevier B.V. All rights reserved.
1. Introduction In the past decades, sulforaphane (SF) has attracted substantial attention thanks to its antioxidant, anti-inflammatory, and anticarcinogenic properties [1]. SF is an isothiocyanate derived from glucoraphanin (GR) and has been proved to be one of the best natural inducer of phase II enzymes in human; these enzymes can detoxify cancer-causing chemicals thus protect against carcinogens and inflammation [2]. When consumed, the plant tissues and cells are damaged and GR comes into contact with one endogenous enzyme, myrosinase (thioglucoside glucohydrolase, EC 3.2.1.147) [3]; then GR is hydrolyzed directly into SF under certain conditions. GR richly exists in Brassica oleracea vegetables, such as broccoli, cabbage, cauliflower [4], among which, seeds from broccoli cultivars are the most likely source for GR as compared with other parts of cruciferous vegetables [1,5,6]. Many studies have aimed at improving the extraction efficiency of sulforaphane from different plant resources. On the one hand, there is no doubt that the hydrolysis reaction conditions like pH, ⁎ Corresponding authors. E-mail addresses: [email protected] (D. Li), [email protected] (L. Wang).
epithiospecifier protein (ESP) activity, Fe2+, ascorbic acid, time, and temperature can influence conversion efficiency of GR to SF [1,4,7]. On the other hand, since it requires a longer time for the conventional extraction method, some assisted extraction techniques with higher extraction efficiency should be explored to enhance the yield and bioactivity of desired products [8]. Recently, several assisted methods have been employed to improve the extraction efficiency of bioactive compounds from plant seeds, including gamma irradiation, ultrasonication, microwave, hydrostatic pressure, and dynamic pressure homogenization [8–11]. Teh and Birch [12] found that ultrasonic treatment (UA) at room temperature increased the extraction content of polyphenol from hemp, flax and canola seeds cake. Chemat et al. [13] used a microwave-assisted method (MW) to extract the carvone and limonene from caraway seeds, and the cell destruction of seeds was observed from the scanning electron micrographs. Prasad et al. [14] also found that ultrasonication and high-pressure extractions had a potential to achieve high extraction efficiency from plant tissues. High hydrostatic pressure technique (HHP), with pressure ranging from 100 to 1000 MPa, has been widely used to enhance mass transfer [15,16] through increasing cell permeability as well as secondary metabolite diffusion [8]. Briones-Labarca et al. [8] used both ultrasonic extraction
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and high hydrostatic pressure methods to improve the release of bioactive compounds from papaya seeds. Extraction of antioxidant and anticancer compounds from plant seeds is a challenge because of the presence of rigid shell of the seeds and the difficulty in reducing the particle size [17,18]. Compared with traditional extraction techniques, the above methods each has their own advantages like time-saving, high efficiency, and high extraction yields. The UV, MW, and HPP methods could enhance the mass transfer and release the bioactive compounds from plant tissues, either by cavitation force [12], rapid rise of temperature in plant cells [19], or high hydrostatic pressure [15], respectively. Moreover, one dynamic high pressure technique has been developed to assist in extracting intracellular components from food and food waste [20,21]. Unlike hydrostatic pressure, high-pressure homogenization (HPH, 20-200 MPa) or microfluidization forces fluid to continuously flow through a homogenizing valve using a positive displacement pump [18,22]. HPH has been reported to cause particle size reduction and was used for pharmaceuticals, food, and other materials, manufacturing industries [23]. HPH can disrupt cellular structures of plant seeds and help release the bioactive compounds into the extracellular environment. However, reports focusing on the application of high-pressure homogenization in sulforaphane extraction are limited. During HPH process, high velocity micro-streams can result in high shear stress when passing through the narrow channel of the chamber [24]. The subsequent resultant cavitation, shear, turbulence and shock waves could lead to the disruption of suspended particles and the release of cellular products [20,22,25]. Furthermore, the ultra-high pressure up to 200 MPa may also impose the similar HHP effect on the seeds tissue, thus increasing the access of solvent to cellular materials [21]. Therefore, the high-pressure homogenization has the potential of improving the extraction efficiency of sulforaphane from plant seeds. The present work was to study the feasibility of using high-pressure homogenization as an assisted method to increase the extraction yield of sulforaphane from broccoli seeds. The particle size and distribution of broccoli seeds after high-pressure homogenization process with different pressures and passes were analyzed. The changes in the morphological structure were also studied using scanning electron micrographs (SEM). Particular interest was given to the relationship between the sulforaphane yield and the particle size. 2. Materials and methods 2.1. Materials and chemicals Fresh Brassica oleracea (SBC2-4AK) seeds were acquired from Todds Seeds (Novi, MI). Following chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO): sulforaphane standard at ≥98% purity, HPLC-grade acetonitrile (ACN), methanol, deionized water, analytical grade (AG) ethanol, AG ethyl acetate, AG hexane, AG anhydrous sodium sulfate, and phosphate buffered saline (PBS). 2.2. Extraction methods 2.2.1. High speed shearing pretreatment (HSS) High speed shearing was used in advance prior to HPH. In this procedure, 10 mM phosphate buffered saline (PBS) solution was prepared by dissolving 1 pack PBS powder in 5 l of distilled water at 25 °C. Broccoli seeds (4 g) were immersed into the PBS (200 mL) solutions at room temperature (pH = 7.2) for 30 min to soften the hard shell of seeds. Seeds were homogenized with a high speed shear mixer (T25 Ultraturrax, IKA Works, Inc. USA) at 11000 rpm for 30 s. This HSS procedure was followed by the high-pressure homogenization procedure described below. 2.2.2. High-pressure homogenization (HPH) HSS treated samples were then transferred to a high-pressure microfluidizer (M110-Y, Microfluidics Corp. USA) equipped with a Z
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type chamber (H10Z, 100 μm), and homogenized with the different pressures of 3000, 5000, 8000, 10,000, 13,000, 18,000, and 23,000 psi (20-160 MPa) and passes of 1, 2, 3, 4, and 5, respectively. In this study, HPH Samples were abbreviated and named as “3000-1”, which means the samples were treated under the pressure of 3000 psi and 1 pass, etc. 2.2.3. Seeds extraction In this work, broccoli seed treated by high speed shearing (HSS) but without HPH was defined as the control sample. The control and HPH samples were allowed to autolyze for 30 min or 8 h at 25 °C. After autolyzing, the suspension was first vacuum-filtered, and then the wet cake of broccoli seeds was washed and extracted two times with 200 mL of ethyl acetate, which was combined with the former filtrate. The ethyl acetate layers were separated and dried at 35 °C under vacuum in a rotary evaporator Büchi R II (Büchi, Switzerland). The residue was dissolved in 100 mL 10% ethanol in water (v/v) and washed two times with equal volumes of hexane to remove nonpolar contaminants. The ethyl acetate layers were pooled, dried over anhydrous sodium, and dried at 35 °C under vacuum in a rotary evaporator. The residue was dissolved in 25 mL methanol solvent and was then filtered through a 0.45 μm membrane filter prior to injection into HPLC. 2.3. Analysis methods 2.3.1. High performance liquid chromatography (HPLC) Sulforaphane was analyzed with an Agilent Infinity 1260 HPLC (USA), equipped with an Agilent 1260 Quat pump and an Acclaim C30 column (Thermo Fisher Scientific Inc. Waltham, MA), 150 mm × 4.6 mm, 3 μm. The solvent system consists of 20% acetonitrile in water, and then changes linearly over 10 min to 60% acetonitrile, and maintained 100% acetonitrile for 2 min to purge the column. Column oven temperature was set at 30 °C. The flow rate was 1 mL/min, and 10 μL portions were injected into the column. Sulforaphane was detected by UV 254 nm. 2.3.2. Standard curve preparation of sulforaphane A stock solution was prepared with 5 mg of sulforaphane reference standard, which was dissolved and diluted in 5 mL acetonitrile. The desired volumes of the standard stock solution of sulforaphane were pipetted into 1.5 mL Eppendorf Tubes and diluted with 1 mL acetonitrile. The final concentrations of sulforaphane were in the range of 15.625–1000 μg/mL. A 10 μL portion of each solution was subjected to HPLC-UV in 6 replicates, and a cali1bration curve was made. Each solution was injected in duplicate. Peak areas were recorded for all the solutions. Quantification was carried out on the basis of the external standard method. 2.4. Particle size measurements The mean particle sizes of the HPH treated broccoli seeds with different pressures and passes were determined using an LS-13-320 laser diffraction particle size analyzer (Backman Coulter Inc., Brea, CA) with the universal liquid module. The mean of the particle size was calculated from the triplicates. The particle size distribution was also recorded. 2.5. Scanning electron microscopy The morphology of broccoli seeds treated with high-pressure homogenization after vacuum drying was investigated using scanning electron microscope (SEM, JSM-6500F, JEOL). Samples were mounted with double-sided carbon adhesive tabs on aluminum stubs, and sputter coated with gold‑palladium (60%-40%). The sample patterns were observed at an accelerating voltage of 2 or 5 kV.
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2.6. Statistical analysis All experiments were carried out in triplicate and the results were expressed as mean ± standard deviation. SPSS 17.0 software (SPSS Inc., Chicago, USA) was used for analysis of variance (ANOVA) and significant differences were analyzed using Duncan's multiple range tests. A 0.95 confidence level (p b 0.05) was used to determine if a significant difference existed between two mean values.
particle size; and there was no difference among the particle size of samples 13,000 psi with different 3, 4, and 5 passes. Few intact cells would be left in the suspension of broccolis seeds when the actual particle size was reduced to around and smaller than the average cell size, which can be observed from the following morphological analysis. The disruption of big particles can result in the increase in specific surface area [11], which means more content of components (GRs) and endogenous enzymes will be released from the cells and compartments.
3. Results and discussion
3.2. Particle size distribution (PSD)
3.1. Particle size reduction
The particle size distribution (PSD) of some representative broccoli seeds samples were represented in Fig. 1. The broccoli seeds of control sample presented a bimodal size distribution profile ranging from 1 to 1000 μm. Broccoli seeds contain two components: the outside shell and the inside kernel. Based on the observation of wet cake of samples after the vacuum filtration (Fig. 2), the first peak of control sample was supposed to represent the size of broccoli kernel (around 10 μm) while the second one referred to the broccoli shells (around 400 μm). During the homogenization process, the broad distribution profile of particle size gradually became narrower. With increasing processing pressure and pass, the bimodal distribution model of particle size was finally converted to single peaked, thus producing the desired results, including uniform particle and size reduction. In order to explain this observation, a series of images of samples before and after homogenization with various conditions were analyzed. The wet cake images of broccoli seeds are shown in Fig. 2. With the increase of homogenization pressure, a more uniform particle size of HPH samples could be observed; the bicolor of the wet cake was gradually changing to monotonous one. Therefore, we can speculate that the particle size reduction at the beginning of homogenization was mainly due to the tissues crush of shell parts. During the homogenization process, not only the high shear force in the small chamber channel can tear up the tissue rupture [18], but the dynamic pressure on the cellular tissue also impose the similar effect with hydrostatic pressure on the seeds tissue, which may increase the mass transfer of the solvent into materials and the soluble constituents (GR and myrosinase) into solvent [15].
A preliminary study was aimed to detect whether homogenization process had an effect on the size reduction of broccoli seed since processing broccoli seeds with homogenizer is a great challenge because of the presence of rigid shell of the seeds [14,18]. Before this homogenization experiment, the broccoli seeds were immersed in the PBS solution to soften the broccoli seeds. Subsequently, the high speed shearing was used to reduce the particle size of broccoli seeds in advance before going through the homogenizer. It was also reported that the temperature increase during homogenization also helped soften the cell walls and then facilitate the cell disruption [18]. The particle sizes of the broccoli seeds treated with high-pressure homogenization under different pressures (3000–23,000 psi) and passes (1–5 passes) were listed in Table 1. HSS process could effectively break the whole broccoli seeds to small particles of 357 μm. In general, the mean size of broccoli seeds was greatly reduced after HPH pretreatment. The results also showed that HPH combined with high speed shearing pre-treatment achieved the desired level of particle size reduction. Compared with the control sample, the particle size of sample 3000-1 (homogenization with 3000 psi and 1 pass) was reduced by 2 times to 170 μm. Microfluidizer processor is a lab machine that provides high shear rates to maximize the energy-per-unit fluid volume to produce uniform micron particles [26,27]. When employed for cell disruption, the high-pressure homogenization process involves mainly two key factors: pressure and pass [18,22,25]. Table 1 showed that pressure could exert more significant influence on the particle size reduction than the pass did. Even though the particle sizes of samples 3000 psi were gradually reduced from 170 to 111 μm with the increase in pass from 1 to 5, the particle size of control sample can be directly reduced to 115 μm at 5000 psi for only one pass. In the meantime, the size of sample 5000-4 is almost the same with sample 8000-1, and 8000-2 with 13,000-1. On the other hand, the influence of pressure on the particle size declined with increasing number of passes, especially at higher pressure conditions. For example, there is no difference among the particle sizes of sample 13,000-2, 18,000-2, and 23,000-2. Similarly, the effect of the pass on the particle size also varied with the pressure powered by the pump. For all pressure levels, the particle size can be reduced to some extent when the process passes were increased from 1 to 2 or 3. Nevertheless, more passes did not necessarily result in smaller
3.3. Linearity of standard curve The calibration curve on standard sulforaphane solutions showed that there was good linearity over the sulforaphane concentration range from 15.625 to 1000 μg/mL (Fig. 3). And the linear regression analysis of the peak area response (y) versus the theoretical concentration (x) gave the following equation: y = 1.577 × −2.885, r2 = 0.9999. The system precision was analyzed by assaying 6 injections of standard solution and calculating the relative standard deviation (RSD) of the peak area response [28]. The RSD (%) for standard sulforaphane was 1.98. The sulforaphane peak in each broccoli seed extract chromatogram was identified based on the retention time of the standard solution. The similarity of chromatograms between the standard and
Table 1 Particle size (Mean size) of broccoli seeds treated by high-pressure homogenization with different pressure and passes*. Z-Avg (d μm) Pressure (psi)
Pass 1
Pass 2
Pass 3
Pass 4
Pass 5
Control 3000 5000 8000 10,000 13,000 18,000 23,000
357.0 ± 1.9a 170.6 ± 1.7b, A 115.3 ± 1.4c, A 82.8 ± 0.3d, A 76.6 ± 1.0e, A 64.3 ± 0.4f, A 60.7 ± 0.9g, A 52.1 ± 0.5h, A
125.4 ± 1.2a, B 85.1 ± 1.7b, B 64.3 ± 0.9c, B 58.2 ± 0.6d, B 44.0 ± 0.4e, B 43.6 ± 1.9e, B 44.1 ± 1.2e, B
122.5 ± 1.0a, B 61.1 ± 0.6b, C 52.0 ± 0.9c, C 48.3 ± 0.6d, C 36.3 ± 0.6e, C 38.2 ± 0.5e, C 37.1 ± 1.3e, C
112.4 ± 2.0a, C 60.0 ± 1.5b, CD 45.0 ± 0.8c, D 43.2 ± 0.8c, D 36.1 ± 0.5d, C 35.0 ± 0.7d, D 31.1 ± 1.7e, D
111 ± 0.2a, C 57 ± 1.4b, D 42 ± 1.5c, E 44 ± 0.7c, D 37 ± 1.7d, C 33.6 ± 1.3d, D 29 ± 1.9e, D
*, Different lowercase and capital letters within the same column differ significantly (p b 0.05). Values represent mean ± S.D., n = 3.
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Fig. 1. Particle size distribution of high-pressure homogenization treated broccoli seeds. Samples 3000-1 means the broccoli seeds were treated by HPH at 3000 psi and 1 pass, etc.
sample was used to evaluate the suitability of this HPLC method. The HPLC chromatograms of the sulforaphane standards (A) of the theoretical concentration of 500 μg/mL and broccoli seeds extracts of control sample (B) and 5000-5 (C) are shown in Fig. 4. Both the standard and sample presented a single peak at 5.1 min, with no interfering peaks on either side of the target peak. These results indicate that the HPLC method can be used for the quantitative analysis of sulforaphane in broccoli seeds, which is similar to the findings confirmed by Liang et al. [29]. 3.4. Extraction yield (EY) The sulforaphane extracted with ethyl acetate for each sample was quantitated by HPLC method. The HPLC peak area of sulforaphane extract from different broccoli seed samples were recorded and analyzed. The extraction yield was then calculated and expressed as the content (μg) of sulforaphane/weight (g) of raw broccoli seeds. The fresh weight (FW) was used because it gives a better idea of the content really ingested. Table 2 listed the sulforaphane contents in broccoli seeds with homogenization extraction method.
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Fig. 3. The standard curve obtained from sulforaphane analysis by HPLC.
In this study, the effect of high-pressure homogenization with different pressures and passes on the sulforaphane extraction was investigated. The results demonstrated that HPH could be used as an assisted method to help extract sulforaphane from broccoli seeds. Table 2 showed that the extraction yield was greatly increased after high-pressure homogenization. After gentle homogenization, the sulforaphane content of 3000-1 has more than doubled from 549 to 1481 μg/g than the control one. The highest content (2199 μg/g) of sulforaphane was found in sample 5000-5, which was three-fold more than the control one without HPH process. Furthermore, the pressure and passes applied also affected the final extraction yield. Regardless of passes, the extraction yield increase with increasing pressure levels and the mean sulforaphane content of samples under 3000, 5000, and 8000 psi were 1606, 1900, and 1966, respectively. However, the similarity with the effect of pressure on the particle size, higher pressure (13,000, 18,000, 23,000 psi) did not result in a significant increase in yield. At the meantime, the extraction yield can be always increased by increasing the process pass. For instance, the sulforaphane content of sample 3000-5, 5000-5, and 8000-5 were increased by 15.6%, 26.2%, and 13.7% compared with the sample 3000-1, 5000-1, and 8000-1, respectively.
Fig. 2. Wet cake images of HPH broccoli seeds after vacuum filtration process. A-F, Control, 3000-1, 5000-1, 8000-1, 13000-1, 23000-1. Samples 3000-1 means the broccoli seeds were treated by HPH at 3000 psi and 1 pass, etc.
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Fig. 4. The HPLC Chromatograms of the sulforaphane standard (A) of theoretical concentration of 500 μg/mL, broccoli seeds extract of Control (B), and sample 5000-5 (C). Conditions described in HPLC section.
Particular interest was paid to the relationship between the extraction yield and the particle size. The particle size changes are in agreement with the results of extraction yield at the lower pressure homogenization conditions (3000 psi). It is a really interesting finding that the particle size of sample 3000-1 was decreased two times and, at the same time, the sulforaphane extraction yield doubled the content of control sample. However, after this, the extraction yield did not increase at the same pattern with decreasing particle size. For example, the particle size of 23,000-5 has decreased up to 10 times (Table 1) while the highest extraction yield of sample 5000-5 was just 3 times more than the control one. This is because particle size could be reduced when seed tissues were broken down under the shearing and tearing effect, and cell disruption happened during this homogenization process [11]. Ideally, once the particle size of broccoli seeds was reduced to the point that it equals to the mean cell size, the target components contained in all the separated cells will be quite easy to extract. Beyond this point, any increase in pressure or pass will result in all the cell walls Table 2 Sulforaphane contents in broccoli seeds treated with or without high-pressure homogenization.⁎ Samples
Peak area
Peak time (min)
Yield (μg/g)
Control-30 min
135.7
5.04
549.2⁎
Control-8 h 3000–1 3000–3 3000–5 5000–1 5000–3 5000–5 8000–1 8000–3 8000–5 13,000–1 18,000–1 23,000–1
168.6 370.9 407.4 429.4 436.8 441.0 542.1 476.5 461.2 552.0 527.4 496.4 486.2
5.10 5.11 5.11 5.10 5.10 5.10 5.12 5.10 5.14 5.10 5.10 5.07 5.11
679.6 1481.4 1626.0 1713.2 1742.6 1759.2 2199.1 1899.9 1839.9 2159.9 1901.6 1978.8 1938.4
1606.9⁎⁎
1900.3
1966.5
⁎Two control samples treated with shearing process but without HPH were autolyzed for 30 min and 8 h, respectively. 3000–1, Sulforaphane extract of broccoli seeds treated at 3000 psi and 1 pass, etc. ⁎ Values represent the mean of three replicates per accession. ⁎⁎ Values represent the mean of same pressure level.
being broken and cells disrupted. It is easy to understand that all GRs and endogenous enzyme will be released to the reaction system and the highest yield of sulforaphane will become a matter of course under the selected optimum reaction conditions. Thus, extremely high pressures are helpful to reduce the particle size but not necessary to assist the sulforaphane extraction in terms of high-pressure homogenization. However, this does not prevent high-pressure homogenization from being a potential pretreatment of sulforaphane extraction. Here comes to another issue, what is the relationship between the mean cell size and the particle size of 5000-5 with highest sulforaphane yield? This is a key issue to the understanding of the extraction mechanism. To answer this question and to see what effect of dynamic pressure had on the cell walls and cells, the treated broccoli seeds were investigated under scanning electron microscope. 3.5. Morphology and structure We employed SEM to investigate the internal structural changes to broccoli seeds after the homogenization process. The effect of highpressure homogenization on the cell walls and cell destruction could be seen on SEM (500×) micrographs (Fig. 5). Before HPH, the broccoli seeds were broken to small particles (357 μm). The main part of this control sample seed tissues seemed remaining unaffected and most of the cells remained together without disruption. Smooth surface was also observed (Fig. 5A). After the homogenization process, broccoli cells were gradually damaged to different degrees; rough surface and cell disruption can be clearly observed in Fig. 5. Seeds tissues were disintegrated, disrupted, and randomly reassembled to form new smaller cell fragments. Furthermore, some large agglomerates with rough surface were observed. HPH even turned broccoli into flat thin chip-like structure (Fig. 5G); some gels-like cells are highly distorted and tightly aggregated (Fig. 5E, F). We noticed that there were no intact cells left in the system for sample 8000-5 (Fig. 5G), 13,000-1 (Fig. 5H), and 23,000-1 (Fig. 5I). This observation is similar to the report that the original complexes of polymers were totally broken into micro-fragments by homogenization, which also resulted in the irreversible disruption and aggregation of polymers [30]. There is no much difference between the samples treated with higher pressure (13,000 and 23,000 psi).
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Fig. 5. Scanning electron micrographs of broccoli seeds of control (A), 3000-1 (B), 3000-5 (C), 5000-1 (D), 5000-5 (E), 8000-1 (F), 8000-5 (G), 13000-1 (H), and 23000-1 (I) (10μm, 2 kV, 500× magnification).
Besides, we have scaled and measured the cell sizes from these SEM images by using the public software ImageJ 1.42q (National Institute of Health, USA) and found that the cell size of broccoli seeds was around 10-70 μm and the mean cell size was around 20-30 μm (data not shown). Considering both the sample 5000-5 (mean size, 57 μm) rather than other samples had the highest sulforaphane content (Table 2) and a few cells of sample 5000-5 (Fig. 5E) still remained their initial cell shape, we speculated that the particle size of samples do not have to be smaller than the mean cell size to favor the release of GR and endogenous enzymes. At 2000 and 5000× magnification, we also observed clear cell rupture (Fig. 6A), breakage at the edge of cell walls (Fig. 6B) and some
pores through the cell (Fig. 6C). High-pressure homogenization was reported using combined forces of shear, cavitation, and ultra-high pressures to process the samples [25]. These observations confirmed that the broccoli seeds underwent violent shearing and tearing during HSS and HPH [11]. Thus, the findings in this study suggested two extraction mechanisms of HPH: one is that homogenization reduced the particle size, increased the surface area of particles, and facilitated the contact between GR and endogenous enzymes; and the other involved the improvement of diffusion of the essential solvent (PBS solution) from the surface to the inside of the granular walls under the high pressure. All these influences could enhance the rate of mass transfer of cell constituents
Fig. 6. Scanning electron micrographs of broccoli seeds of sample 5000-3. Cell disruption, cell wall breakage, and pores were found in A, B, and C, respectively. (5 kV, 2000 and 5000× magnification).
Please cite this article as: J. Xing, Y. Cheng, P. Chen, et al., Effect of high-pressure homogenization on the extraction of sulforaphane from broccoli (Brassica ole..., Powder Technol., https://doi.org/10.1016/j.powtec.2018.12.010
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J. Xing et al. / Powder Technology xxx (2018) xxx
through the cell walls, which means more content of components and endogenous enzymes will be released from the cells. 4. Conclusions This study employed the high-pressure homogenization technique to assist in extraction of sulforaphane from broccoli seeds. High-pressure homogenization combined with high speed shear provided a better way of pretreating the broccoli seeds to smaller particle size than the conventional method. Varying degrees of size reduction were achieved by HPH with different pressures and passes. The analysis of the relationship between particle size and sulforaphane extraction yield shows that the sulforaphane content did not increase continuously with increasing pressure. In fact, pressure does not have to be very high (b8000 psi, 55 MPa) to successfully maximize the sulforaphane extraction. HPH also changed the original smooth surface to small flake-like structure and fragments. Cell rupture, cell walls breakage, and cell poration were observed in SEM images. The effect of HPH on the extraction was supposed duo to two mechanisms: cell disruption caused by high mechanical force and enhanced mass transfer rates across granular walls under high dynamic pressure. Both facilitated the contact between GR and myrosinase, which increase the sulforaphane yield from broccoli seeds. Thus in this study, high-pressure homogenization treatment showed potential application in extraction of sulforaphane from broccoli seeds. Acknowledgments This research was supported by National Natural Science Foundation of China (31771896). References [1] H. Liang, Q. Yuan, Natural sulforaphane as a functional chemopreventive agent: including a review of isolation, purification and analysis methods, Crit. Rev. Biotechnol. 32 (2012) 218–234. [2] H. Liang, C. Li, Q. Yuan, F. Vriesekoop, Application of high-speed countercurrent chromatography for the isolation of sulforaphane from broccoli seed meal, J. Agric. Food Chem. 56 (2008) 7746–7749. [3] J.W. Fahey, W.D. Holtzclaw, S.L. Wehage, K.L. Wade, K.K. Stephenson, P. Talalay, Sulforaphane bioavailability from glucoraphanin-rich broccoli: control by active endogenous myrosinase, PLoS One 10 (2015), e0140963. . [4] Z.-x. Gu, Q.-h. Guo, Y.-j. Gu, Factors influencing glucoraphanin and sulforaphane formation in brassica plants: a review, Integr. Agric. 11 (2012) 1804–1816. [5] H. Liang, Q. Yuan, Q. Xiao, Purification of sulforaphane from Brassica oleracea seed meal using low-pressure column chromatography, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 828 (2005) 91–96. [6] L. Zhansheng, L. Yumei, Z.Y. Fang, L. Mu Yang, M. Zrfg, Y.Y. Zhang, L.H. Honghao, Development and identification of anti-cancer component of sulforaphane in developmental stages of broccoli (Brassica oleracea var. italica L.), J. Food Nutr. Res. 4 (2016) 490–497. [7] C. Perez, H. Barrientos, J. Roman, A. Mahn, Optimization of a blanching step to maximize sulforaphane synthesis in broccoli florets, Food Chem. 145 (2014) 264–271. [8] V. Briones-Labarca, M. Plaza-Morales, C. Giovagnoli-Vicuña, F. Jamett, High hydrostatic pressure and ultrasound extractions of antioxidant compounds, sulforaphane and fatty acids from Chilean papaya (Vasconcellea pubescens) seeds: Effects of extraction conditions and methods, LWT Food Sci. Technol. 60 (2015) 525–534.
[9] V. Briones-Labarca, C. Muñoz, H. Maureira, Effect of high hydrostatic pressure on antioxidant capacity, mineral and starch bioaccessibility of a non conventional food: Prosopis chilensis seed, Food Res. Int. 44 (2011) 875–883. [10] R.H. Chen, J.R. Huang, M.L. Tsai, L.Z. Tseng, C.H. Hsu, Differences in degradation kinetics for sonolysis, microfluidization and shearing treatments of chitosan, Polym. Int. 60 (2011) 897–902. [11] X. Hua, S. Xu, M. Wang, Y. Chen, H. Yang, R. Yang, Effects of high-speed homogenization and high-pressure homogenization on structure of tomato residue fibers, Food Chem. 232 (2017) 443–449. [12] S.S. Teh, E.J. Birch, Effect of ultrasonic treatment on the polyphenol content and antioxidant capacity of extract from defatted hemp, flax and canola seed cakes, Ultrason. Sonochem. 21 (2014) 346–353. [13] S. Chemat, H. Aït-Amar, A. Lagha, D.C. Esveld, Microwave-assisted extraction kinetics of terpenes from caraway seeds, Chem. Eng. Process. Process Intensif. 44 (2005) 1320–1326. [14] K.N. Prasad, B.A.O. Yang, M. Zhao, N. Ruenroengklin, Y. Jiang, Application of ultrasonication or high-pressure extraction of flavonoids from litchi fruit pericarp, J. Food Process Eng. 32 (2009) 828–843. [15] H.-W. Huang, C.-P. Hsu, B.B. Yang, C.-Y. Wang, Advances in the extraction of natural ingredients by high pressure extraction technology, Trends Food Sci. Technol. 33 (2013) 54–62. [16] Z. Shouqin, Z. Junjie, W. Changzhen, Novel high pressure extraction technology, Int. J. Pharm. 278 (2004) 471–474. [17] K.N. Prasad, J. Hao, J. Shi, T. Liu, J. Li, X. Wei, S. Qiu, S. Xue, Y. Jiang, Antioxidant and anticancer activities of high pressure-assisted extract of longan (Dimocarpus longan Lour.) fruit pericarp, Innovative Food Sci. Emerg. Technol. 10 (2009) 413–419. [18] N. Samarasinghe, S. Fernando, R. Lacey, W.B. Faulkner, Algal cell rupture using high pressure homogenization as a prelude to oil extraction, Renew. Energy 48 (2012) 300–308. [19] W. Sookjitsumran, S. Devahastin, A.S. Mujumdar, N. Chiewchan, Comparative evaluation of microwave-assisted extraction and preheated solvent extraction of bioactive compounds from a plant material: a case study with cabbages, Innovative Food Sci. Emerg. Technol. 51 (2016) 2440–2449. [20] P. Comuzzo, S. Calligaris, L. Iacumin, F. Ginaldi, S. Voce, R. Zironi, Application of multi-pass high pressure homogenization under variable temperature regimes to induce autolysis of wine yeasts, Food Chem. 224 (2017) 105–113. [21] X. Huang, Z. Tu, Y. Jiang, H. Xiao, Q. Zhang, H. Wang, Dynamic high pressure microfluidization-assisted extraction and antioxidant activities of lentinan, Int. J. Biol. Macromol. 51 (2012) 926–932. [22] C.H. Karacam, S. Sahin, M.H. Oztop, Effect of high pressure homogenization (microfluidization) on the quality of Ottoman Strawberry (F. Ananassa) juice, LWT Food Sci. Technol. 64 (2015) 932–937. [23] X. Zhu, Y. Cheng, P. Chen, P. Peng, S. Liu, D. Li, R. Ruan, Effect of alkaline and highpressure homogenization on the extraction of phenolic acids from potato peels, Innovative Food Sci. Emerg. Technol. 37 (2016) 91–97. [24] B.C. Porto, P.E. Augusto, A. Terekhov, B.R. Hamaker, M. Cristianini, Effect of dynamic high pressure on technological properties of cashew tree gum (Anacardium occidentale L.), Carbohydr. Polym. 129 (2015) 187–193. [25] W. Liu, J. Liu, C. Liu, Y. Zhong, W. Liu, J. Wan, Activation and conformational changes of mushroom polyphenoloxidase by high pressure microfluidization treatment, Innovative Food Sci. Emerg. Technol. 10 (2009) 142–147. [26] C. Baldwin, C.W. Roblnsonf, Disruption of Saccharomyces cerevisiae using enzymatic lysis combined with high-pressure homogenizatio, Biotechnol. Tech. 4 (1990) 329–334. [27] A. Carlson, M. Signs, L. Liermann, R. Boor, K.J. Jem, Mechanical disruption of Escherichia coli for plasmid recovery, Biotechnol. Bioeng. 48 (1995) 303–315. [28] P. Kuang, D. Song, Q. Yuan, R. Yi, X. Lv, H. Liang, Separation and purification of sulforaphene from radish seeds using macroporous resin and preparative high-performance liquid chromatography, Food Chem. 136 (2013) 342–347. [29] H. Liang, C. Li, Q. Yuan, F. Vriesekoop, Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography, J. Agric. Food Chem. 55 (2007) 8047–8053. [30] J.L. Hu, S.P. Nie, M.Y. Xie, High pressure homogenization increases antioxidant capacity and short-chain fatty acid yield of polysaccharide from seeds of Plantago asiatica L, Food Chem. 138 (2013) 2338–2345.
Please cite this article as: J. Xing, Y. Cheng, P. Chen, et al., Effect of high-pressure homogenization on the extraction of sulforaphane from broccoli (Brassica ole..., Powder Technol., https://doi.org/10.1016/j.powtec.2018.12.010