Article pubs.acs.org/JAFC
Analysis of Apoptosis in Ultraviolet-Induced Sea Cucumber (Stichopus japonicus) Melting Using Terminal DeoxynucleotidylTransferase-Mediated dUTP Nick End-Labeling Assay and Cleaved Caspase‑3 Immunohistochemistry Jing-Feng Yang,† Rong-Chun Gao,† Hai-Tao Wu,† Peng-Fei Li,† Xian-Shu Hu,† Da-Yong Zhou,*,† Bei-Wei Zhu,*,† and Yi-Cheng Su‡ †
School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian 116034, P. R. China ‡ Seafood Research and Education Center, Oregon State University, Astoria, Oregon 97103, United States ABSTRACT: The sea cucumber body wall melting phenomenon occurs under certain circumstances, and the mechanism of this phenomenon remains unclear. This study investigated the apoptosis in the ultraviolet (UV)-induced sea cucumber melting phenomenon. Fresh sea cucumbers (Stichopus japonicus) were exposed to UV radiation for half an hour at an intensity of 0.056 mW/cm2 and then held at room temperature for melting development. The samples were histologically processed into formalinfixed paraffin-embedded tissues. The apoptosis of samples was analyzed with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and cleaved caspase-3 immunohistochemistry. The emergence of TUNEL-positive cells speeds up between 0.5 and 2 h after UV irradiation. Cleaved caspase-3 positive cells were obviously detected in sample tissues immediately after the UV irradiation. These results demonstrated that sea cucumber melting induced by UV irradiation was triggered by the activation of caspase-3 followed by DNA fragmentation in sea cucumber tissue, which was attributed to apoptosis but was not a consequence of autolysis activity. KEYWORDS: apoptosis, autolysis, melting, TUNEL, caspase-3
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healthy sea cucumbers.2 Although these enzymes have been reported to be closely related to apoptosis,9−12 the mechanism of their involvement in the body wall melting phenomenon of sea cucumbers remains unclear. Through apoptosis, a number of changes in cells, including blebbing, shrinkage, nucleic acid fragmentation, chromatin condensation, and chromosomal DNA fragmentation, may occur13 and cause the death of cells. Analysis of apoptosis in sea cucumbers can help in the understanding of the mechanism of its body wall melting phenomenon. In this study, we investigated signs of apoptosis, such as DNA fragmentation and the activation of caspase-3, in sea cucumbers with body wall melting phenomenon. This research contributes a new understanding of the sea cucumber body wall melting phenomenon, which was previously believed to be autolysis.
INTRODUCTION Sea cucumbers are echinoderms from the class Holothuroidea and have a long history of being regarded as a seafood delicacy in East Asia, especially in China and Japan.1 Sea cucumber is susceptible to environmental changes and factors including temperature, salt concentration, nutrient deficiency, exposure to sunlight, ultraviolet (UV) irradiation, and mechanical stimulation.2 All these factors may trigger the sea cucumber to initiate the “melting” body wall phenomenon, which is followed by death. This phenomenon is a major concern for sea cucumber production, particularly during transportation and processing.3 The body wall melting phenomenon of sea cucumber under certain circumstances is generally believed to be associated with autolysis,4−6 which is commonly known as self-digestion of cells through the actions of its own enzymes. Autolysis is initiated in injured cells or dying tissue by releasing digestive enzymes from lysosomes into the cytoplasm; this phenomenon is often encountered after the death of marine animals, such as shrimp7 and octopus.8 The autolysis phenomenon typically begins with necrosis, which is a form of traumatic cell death resulting from severe cellular injury. However, the body wall melting phenomenon of the sea cucumber is more likely to be apoptosis (a process of programmed cell death) because the melting phenomenon often occurs when they are still alive. Previous study showed that increased activities of cathepsin Blike and L-like enzymes were observed in the body wall of sea cucumbers developing the melting phenomenon but not in © XXXX American Chemical Society
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MATERIALS AND METHODS
Materials. Sexually mature adult sea cucumbers (Stichopus japonicus, 100−120 g; 15−20 cm total body length) were captured in November along the coastline of Dalian, China. Freshly harvested sea cucumbers were transported to the laboratory within 1 h by ice covering using a bubble chamber. Ethanol, dimethylbenzene potassium dihydrogen phosphate, disodium hydrogen phosphate, tosylchloramide sodium, citrate, trichloroacetic acid, sodium chloride, and Received: July 17, 2015 Revised: October 14, 2015 Accepted: October 14, 2015
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DOI: 10.1021/acs.jafc.5b03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Caspase-3 Activity Determination. Caspase-3 activity was determined using a caspase-3 assay kit (Sigma-Aldrich, St. Louis, MO, US) containing caspase-3 positive control, assay buffer, lysis buffer, and caspase-3 substrate. The activity of caspase-3 was analyzed based on the release of the p-nitroaniline (pNA) moiety through hydrolysis of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3 (one unit of caspase-3 will cleave 1.0 μmol of Ac-DEVDpNA per min at pH 7.4 at 25 °C) following the manufacturer’s instructions. Briefly, sea cucumber intestine (0.3 g wet weight) was homogenized in 0.9 mL of lysis buffer and incubated for 15 min at 4 °C. Then, the sample was centrifuged (Thermo Fisher Scientific, Waltham, MA, US) at 400g for 10 min at 4 °C. The supernatant was transferred to a new tube and centrifuged at 12 000g for 20 min at 4 °C. The supernatant was collected and analyzed immediately. The 5 μL samples of supernatants and caspase-3 positive control were transferred to wells of a microtiter plate, and 1 × assay buffer (85 μL) was added to each well. The reaction was started by adding 10 μL of caspase-3 substrate to each well and mixing gently by shaking. The reaction mixture was incubated at 37 °C for 90 min, and the absorbance was read at 405 nm (Tecan, Männedorf, Switzerland). Results were calculated using a p-nitroaniline calibration curve. TCA-Soluble Peptide Content Determination. The trichloroacetic acid (TCA) soluble peptide content was measured to determine the autolysis in the initial stage of sea cucumber melting induced by UV irradiation. After UV irradiation, the sea cucumber body wall (back and belly parts) sample (30 g) was homogenized and centrifuged at 3000g for 10 min at 4−5 °C. Each sample supernatant was adjusted to 10 mL by adding distilled water followed by mixing with an equal volume of TCA (30%, m/V) to obtain the final concentration of TCA (15%, m/V) in the sample suspension. All mixed samples were held for 20 min at room temperature and centrifuged at 16 500g for 10 min (CF16RX II, HITACHI, Tokyo, Japan) at 4−5 °C. The supernatant was collected for analysis of TCA-soluble peptides by the Lowry method15 using BSA as a standard (Sangon Biotech Co., Ltd. Shanghai, China). Hydroxyproline Content Analysis. The hydroxyproline content in the sample supernatant containing TCA-soluble peptides was determined by mixing of the sample (0.5 mL) with 3 mL of 6 mol/L hydrochloric acid and hydrolyzing at 130 °C for 4 h. The hydrolysis products were allowed to cool, neutralized with NaOH (6 M), and brought to a volume of 10 mL with distilled water. Then, 1 mL of the hydrolyzed sample was mixed with 1 mL of citrate buffer solution and 1 mL of tosylchloramide sodium and blended for 10 min at room temperature. The sample was then mixed with 1 mL of 3.5 M perchloric acid and blended for 10 min at room temperature followed by adding 1 mL of chromogenic reagent; the sample was then incubated in a water bath at 65 °C for 20 min. The chromogenic reagent was prepared by mixing 2 g of p-dimethylaminobenzaldehyde (Tianjin Aoran Fine Chemical Research Institute, Tianjin, China) with 7 mL of perchloric acid (Tianjin Zhengcheng Co., Ltd. Tianjin, China) and 13 mL of isopropanol (Tianjin Kemio Co., Ltd. Tianjin, China). Heated samples were allowed to cool to room temperature, and the absorbency of samples was measured at 560 nm with a microplate reader (Tecan, Männedorf, Switzerland). Cell Counting and Statistical Analysis. We performed quantification of immunolabeled cells with TUNEL and cleaved caspase-3 immunohistochemistry. For each assay, three fields of 0.04 mm2 from each sample were quantified at 400× magnification, and results were reported as means of three determinations. Significant differences (p < 0.01) between means per area and group were determined by one-way analysis of variance using SPSS.
potassium chloride were purchased from Tianjin Damao Co. Ltd. (Tianjin, China). All chemicals used were analytical grade or above. The experiments were performed in compliance with the appropriate laws and institutional guidelines of the Institutional Laboratory Animal Care and Use Committee of The Dalian Medical University. Inducing Sea Cucumber Melting by UV Radiation. Sea cucumbers were randomly divided into seven groups with eight samples in each group. Six groups of samples were exposed to UV radiation (253.7 nm, Cnlight, ZW20S19W, Shenxing, Jiangyin, Jiangsu, China) for 0.5 h at an intensity of 0.056 mW/cm2, and the seventh group without UV irradiation was used as control. An additional group (six samples) without UV irradiation was kept at room temperature for 6 h as a comparison to the control for eliminating potential interference caused by factors such as temperature, salt concentration, and nutrient deficiency. After UV irradiation, the six groups of sea cucumbers were held at 20 °C under a florescent lamp for 0, 30, 60, 120, 240, or 360 min before analysis. Tissue Preparation. Each of the UV-treated sea cucumbers was cut at the end of the cloacal aperture, and the intestine was removed. The belly of the sea cucumber was cut, and the back skin tissue was sampled to tissue bricks (1 cm × 1 cm × 0.5 cm). The tissue bricks were fixed by immersion in 10% neutral formalin in phosphate buffered saline (PBS). After fixation for 24 h, tissues were dehydrated, paraffin embedded, and sectioned for histological experiment. Sections 5 μm thick were heated in a distilled water bath (45 °C), collected on a glass slide, deparaffinised, rehydrated stepwise through an ethanol series, and processed for routine histological analysis.14 The intestines of each group of samples were homogenized immediately after their removal and analyzed for caspase-3 activity. Terminal Deoxynucleotidyl-Transferase-Mediated dUTP Nick End-Labeling (TUNEL) Immunohistochemistry. For in situ detection of DNA fragmentation in paraffin-embedded tissue of sea cucumber, the TUNEL immunohistochemistry analysis was performed using the TUNEL Apoptosis Assay kit (Roche, South San Francisco, CA, US) according to the manufacturer’s instructions. Briefly, tissue slides were deparaffinized and treated with proteinase K (20 μg/mL) for 20 min at 25 °C. Endogenous peroxidase was then inactivated with 3% H2O2 in PBS solution for 5 min. The labeling mixture containing biotinylated dUTP in TdT enzyme buffer was added to sections, and the samples were incubated at 37 °C in a humidity chamber (Yiheng, Tech. Co. Ltd., Shanghai, China) for 1 h. After the enzymatic reaction stopped, sections were rinsed with PBS, covered with antidigoxigenin peroxidase conjugate, and incubated for 30 min at 25 °C in a humidity chamber (Yiheng, Tech. Co. Ltd., Shanghai, China). Then, sections were incubated with 0.05% diaminobenzidine (DAB, Beyotime, Shanghai, China) plus 3% hydrogen peroxide until color development was achieved. Finally, sections were washed and counterstained in hemanoxylin and eosin staining (H&E, Leagene, Beijing, China) for 3 min. Cleaved Caspase-3 Immunohistochemistry. Deparaffinised and rehydrated tissue sample sections were subjected to an antigen retrieval procedure by heating the samples in an oven at 65 °C for 30 min. After cooling for 10 min, sections were rinsed in proper order of dimethylbenzene, ethanol, and distilled water for 5 min followed by incubation in 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase in samples. Antigen retrieval was done by immersion of sections in 0.01 M citrate (Sigma-Aldrich, St. Louis, MO, US) buffer bath (pH 6.0, 92 °C) for 40 min. All heated samples were rinsed with PBS solution for 5 min at room temperature, which was repeated 3 times followed by immersion in 5% bovine serum albumin (BSA, Sangon Biotech, Shanghai, China) at room temperature for 10 min.14 The samples were then incubated with the diluted caspase-3 antibody solution (1/1000 in PBS, Cell Signaling Technology, Inc., Danvers, MA, US) at 4 °C overnight; sections were washed with PBS three times for 5 min each. Sections were adjusted to room temperature, incubated with goat antirabbit IgG-HRP (1/5000 in PBS) (sc-2030, Santa Cruz Biotechnology, Dallas, TX, US) for 30 min at room temperature, and subsequently treated with 0.05% DAB for 10 min at room temperature. Finally, sections were rinsed in distilled water, counterstained with H&E, flushed with water, and dried in air.
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RESULTS AND DISCUSSION Melting of Sea Cucumber Induced by UV Irradiation. The sea cucumber melting phenomenon can be triggered by a variety of factors, such as temperature, salt concentration, nutrient deficiency, exposure to sunlight, UV irradiation, and mechanical stimulation. In this study we developed an UV irradiation model to facilitate study of the melting procedure. B
DOI: 10.1021/acs.jafc.5b03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. Morphological changes of melting sea cucumber induced by exposure to UV radiation (0.056 mW/cm2 for 0.5 h) and held at room temperature: (A) fresh sea cucumber, (B) 1 h after UV exposure, (C) 4 h after UV exposure, (D) 6 h after UV exposure, (E) 26 h after UV irradiation, and (F) 52 h after UV exposure.
Figure 2. In situ detection of DNA fragmentation in sea cucumber cells by TUNEL assay and counterstained by hemanoxylin and eosin staining (H&E). The cross-sectional tissue slice of sea cucumber body wall near the epidermis was examined under an optical microscope (400×): (A) control, (B) 0.5 h after UV irradiation, (C) 1 h after UV irradiation, (D) 2 h after UV irradiation, (E) 4 h after UV irradiation, and (F) 6 h after UV irradiation. TPC, TUNEL positive cell; TNC, TUNEL negative cell. C
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Figure 3. TUNEL assay of the inner tissue slice of sea cucumber body wall counterstained with H&E after UV irradiation. Samples were examined under an optical microscope (400×, A−G): (A) control, (B) 0.5 h after UV irradiation, (C) 1 h after UV irradiation, (D) 2 h after UV irradiation, (E) 4 h after UV irradiation, and (F) 6 h after UV irradiation. TPC, TUNEL positive cell; TNC, TUNEL negative cell. Panel G presents ratios of TUNEL-positive cells to total cells analyzed with TUNEL staining.
stained by H&E, while the TUNEL-positive cells appeared as brown spot because the fragmented nucleus was labeled by biotinylated dUTP. TUNEL-positive cells were clearly detected in UV-irradiated sea cucumbers at various stages of melting development with more and more TUNEL-positive cells being detected in the samples as the melting progressed. However, a few TUNEL-positive cells were detected in the control group. The TUNEL-positive cells detected in the control group could be due to a natural process or the stress occurring under unfavorable growth circumstances when samples were transported to the lab. The TUNEL immunohistochemistry analysis of the inner body wall tissues of UV-irradiated sea cucumber counterstained with H&E is showed in Figure 3. To clarify whether apoptotic death of sea cucumber body wall cell (SBC) was interrelated with the melting phenomenon, the TUNEL-positive cells in UV-irradiated sea cucumbers showing body wall melting were
The melting process of sea cucumbers was less apparent in samples within 6 h after UV irradiation (Figure 1A−F). During the early stage of the melting process, the sea cucumber showed squirming capability in response to touch, and some perceivable morphological change of perisome was observed in sea cucumber 6 h after UV irradiation (Figure 1D). Detection of TUNEL-Positive Cells in Sea Cucumber Body Wall. The apoptosis during sea cucumber melting was first determined by the immunohistochemical detection of DNA strand breaks with TUNEL staining of paraffin-embedded sections. The sea cucumber body wall consists of an epidermis covered by a thin cuticle and a dermis which is composed of an outer pigmented layer; a dense, white inner layer; and an inner circular muscle layer.16−18 Analysis of the results showed that sea cucumber perisome cells mainly amassed in the epidermis but scattered in the inner layer (Figure 2). TUNEL-negative cells appeared as blue spots because the holonomic nucleus was D
DOI: 10.1021/acs.jafc.5b03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 4. Cleaved caspase-3 immunoreactivity assay of UV-irradiated sea cucumber body wall cells counterstained with H&E. Samples were examined under an optical microscope (400×; caspase-3 negative cells (blue spots) and caspase-3 positive cells (brown spots)): (A) control, (B) 0 h after UV irradiation, (C)1 h after UV irradiation, (D) 2 h after UV irradiation, (E) 4 h after UV irradiation, and (F) 6 h after UV irradiation. Panel G presents ratios of cleaved caspase-3 positive cells to total cells analyzed with cleaved caspase-3 immunohistochemistry staining. CPC, cleaved caspase3 positive cell; CNC, cleaved caspase-3 negative cell.
greater than 50% 6 h after exposure to UV radiation. Sea cucumber samples without UV radiation and kept under the same conditions for 6 h were also examined by TUNEL immunohistochemistry analysis as a comparison. The result demonstrated that no TUNEL-positive cells were detected in sea cucumber samples without UV exposure and held at 20 °C for 6 h (data not shown). Internucleosomal fragmentation of DNA into oligonucleotides of 180−200 bp, or multiples thereof, is a classic end point of apoptotic cell death.19 The activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. Therefore, DNA fragmentation revealed by the presence of a multitude of DNA strand breaks is considered to be the gold standard for
quantified and compared with the control samples. The ratios of TUNEL-positive cells to the total cells in the sea cucumbers showing UV-induced body wall melting were remarkably higher than those in samples without UV exposure. As shown in Figure 3F, the incidence of TUNEL-positive cells in samples 6 h after stopping exposure to UV radiation was much higher than those of less melting phenomenon (0.5, 1, 2, and 4 h after stopping exposure to UV) and controls. A significant speedup of TUNEL-positive cells increase (p < 0.01) was observed in samples between 0.5 and 2 h after stopping exposure to UV radiation, which indicates that the apoptotic cell death in the samples occurred rapidly within this period and continued with a slower increase rate thereafter. The ratio of TUNEL-positive cells to total cells in UV-irradiated sea cucumbers increased to E
DOI: 10.1021/acs.jafc.5b03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. Change of caspase-3 activity in sea cucumber intestinal cells after exposure of sea cucumber to UV radiation. *, p < 0.05, indicates significant difference versus control group; **, p < 0.01, indicates significant difference versus control group.
identifying apoptotic cells.20 In this study, DNA fragmentation was observed in the tissue cells of UV-exposed sea cucumbers, but it was hardly seen in any cells of sea cucumbers without UV exposure. This result demonstrates that UV irradiation induced the apoptosis in sea cucumber body wall cells and then triggered the melting phenomenon in the sea cucumbers. Apoptosis thus is suggested to be involved in the development of sea cucumber melting. Cleaved Caspase-3 Immunoreactivity. The cleaved caspase-3 immunoreactivity was carried out to confirm the SBC apoptosis involved in sea cucumber melting. Similar to the observation of TUNEL-positive cells, cleaved caspase-3 positive cells appear as brown spots with faint background in the slice of sea cucumber body wall samples, whereas cleaved caspase-3 negative cells appear as blue spots because the condensed nuclear chromatin is counterstained with H&E. Almost no cleaved caspase-3 positive cells were detected in healthy sea cucumber tissues (Figure 4A), which indicates that there were no apoptosis cells in normal sea cucumber body walls. However, cleaved caspase-3 positive cells were detected in sea cucumber body wall tissues after UV irradiation (Figure 4B−F). Cleaved caspase-3 positive cells showed the strongest phase at 0 h after UV inducing. Thereafter, the ratio of cleaved caspase-3 positive cells decreased, but the ratio of positive cells showed a small increase in the later stages (Figure 4B). Caspase-3 Activity Determination in Sea Cucumber Intestinal Cells. To confirm the role of cleaved caspase-3 immunoreactivity in SBC during sea cucumber melting, changes of caspase-3 activity in sea cucumber intestinal cells during melting stage were determined and compared with those observed from fresh sea cucumbers. A rapid increase in caspase3 activity from 2.4 to 14.5 U/mg was observed in intestinal cells of sea cucumbers immediately after UV irradiation (Figure 5). However, the caspase-3 activity decreased dramatically to 4.2 U/mg 1 h after the UV irraditation and then increased slightly to 6.5 U/mg 4 h after the UV treatment. These results agreed with the results of cleaved caspase-3 immunoreactivity (Figure 4). The rapid increase of caspase-3 activity before the occurrence of TUNEL-positive cells demonstrated that sea cumcumber melting was associated with activation of caspase-3 and following DNA fragmentation. Furthermore, the intestine had less radiation than the body wall during UV exposure. This experiment, which was performed with intestinal tissue, can also test whether a lower degree of UV irradiation could trigger melting phenomenon in sea cucumber. The results showed that
even a lower amount of UV exposure caused the melting of the sea cucumber. Caspases are a group of aspartate-specific cysteine proteases synthesized as precursors which require proteolytic conversion to become active caspases.21,22 Among them, caspase-3 is one of the known effector caspases which, once activated, irreversibly executes cell death through degradation of vital cell proteins and activation of endonucleases.14 Thus, the activation of caspase-3 is considered a hallmark of apoptosis.23 Cleaved caspase-3 is the major performer of apoptotic nuclear changes, and it ultimately leads to low molecular weight DNA fragmentation, which made the TUNEL-detectable.24 Autolysis Assay. Autolysis generally occurs immediately after death of animals with a number of biochemical and enzymatic changes along with microbial activity. It has been reported that autolysis was observed in fish muscle after death and leads to the disintegration of muscle structure.25 Currently, several enzymes (such as cathepsins B, D, and L) have been confirmed to be involved in autolysis.26 However, no particular enzyme can be definitively determined to be the dominant one. Hence, the TCA-soluble peptides content (released from the enzymatic activity) is considered a good index for analysis of the autolysis.7,27 In this study, the TCA-soluble peptides contents in both the back and belly tissues are nearly constant within 6 h after UV exposure (Figure 6). These results indicated that autolysis was not observed within 6 h. On the other hand, both the cleaved caspase-3 immunoreactivity and the TUNEL assays revealed that the apoptosis in sea cucumber
Figure 6. Amount of change of total TCA-soluble peptide extracted from back tissue and belly tissue of sea cucumber body wall from 0 to 6 h after UV exposure. F
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occurred within 2 h after UV exposure. These results indicated that the sea cucumber melting phenomenon induced by UV was triggered by apoptosis but not autolysis. Collagen is the principal component protein in sea cucumber body walls, and the insoluble collagen fiber accounts for 70% of the protein in sea cucumbers.28,29 Proline and hydroxyproline account for one-third of the total amino acids in sea cucumber collagen. Therefore, detection of hydroxyproline in TCAsoluble peptide fraction would indicate the activation of autolysis in sea cucumber melting. As can be seen in Figure 7, the content of dissociative hydroxyproline remained nearly
The authors declare no competing financial interest.
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Figure 7. Amount of change of dissociative hydroxyproline during sea cucumber body wall melting from 0 to 6 h after UV exposure.
constant in TCA-soluble peptides until 6 h after UV exposure during sea cucumber melting. This result demonstrated that the sea cucumber collagen maintained its structure and was not degraded by autolysis in the early stage, which also proved that the autolysis was not involved in the initial stage of sea cucumber melting. In conclusion, both the cleaved caspase-3 immunoreactivity and the TUNEL assays revealed that the sea cucumber melting after UV irradiation was associated with apoptosis in sea cucumber body wall cells. The detection of TCA-soluble peptides and hydroxyproline content increase 6 h after UV exposure in melting sea cucumber tissue also suggested that the apoptosis leads in autolysis. On the basis of this finding, it is concluded that the initiation of the sea cucumber melting phenomenon triggered by UV exposure is attributed to apoptosis but not autolysis.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Authors
*B.-W.Z.: School of Food Science and Technology, Dalian Polytechnic University, No. 1 Qinggongyuan, Ganjingzi District, Dalian 116034, P. R. China. Tel: +86-411-86323262. Fax: +86-411-86323262. E-mail:
[email protected]. *D.-Y.Z.: School of Food Science and Technology, Dalian Polytechnic University, No. 1 Qinggongyuan, Ganjingzi District, Dalian 116034, P. R. China. Tel: +86-411-86323262. Fax: +86-411-86323262. Funding
This work was financed by The National Natural Science Foundation (31301430), Science and technology research projects in Liaoning province (2014205001), and Major in colleges and universities in Liaoning province science and technology platform ([2011]191). G
DOI: 10.1021/acs.jafc.5b03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.jafc.5b03453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
