Tenderness and histochemistry of muscle tissues from Eriocheir sinensis

Tenderness and histochemistry of muscle tissues from Eriocheir sinensis

Journal Pre-proof Tenderness and histochemistry of muscle tissues from Eriocheir sinensis Long Zhang, Wenli Wang, Fen Zhou, Yao Zheng, Xichang Wang PI...

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Journal Pre-proof Tenderness and histochemistry of muscle tissues from Eriocheir sinensis Long Zhang, Wenli Wang, Fen Zhou, Yao Zheng, Xichang Wang PII:

S2212-4292(18)31196-9

DOI:

https://doi.org/10.1016/j.fbio.2019.100479

Reference:

FBIO 100479

To appear in:

Food Bioscience

Received Date: 6 December 2018 Revised Date:

24 October 2019

Accepted Date: 26 October 2019

Please cite this article as: Zhang L., Wang W., Zhou F., Zheng Y. & Wang X., Tenderness and histochemistry of muscle tissues from Eriocheir sinensis, Food Bioscience (2019), doi: https:// doi.org/10.1016/j.fbio.2019.100479. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical abstract

1

Tenderness and Histochemistry of Muscle Tissues from

2

Eriocheir sinensis

3

Long Zhang#, Wenli Wang#, Fen Zhou, Yao Zheng, Xichang Wang*

4

College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, PR

5

China

6

Running Title: Tenderness of Eriocheir sinensis

7 8

# These authors contributed equally to this study.

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*Corresponding author: Xichang Wang

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Address: No.999, Huchenghuan Rd, Nanhui New City, Pudong New District, Shanghai, P.R.

11

China

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Tel: +86 21 61900051; Fax: +86 21 61900054;

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E-mail address: [email protected]

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1

15

Abstract

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Texture is the most important factor influencing consumer satisfaction with crab

17

meat palatability. A valuable micro method using particle size analysis was developed

18

to qualitatively determine cooked meat tenderness of different muscle tissues. The test

19

was successfully used with Chinese mitten crab (Eriocheir sinensis), horse crab

20

(Portunus trituberculatus), manila clam (Ruditapes philippinarum), red swamp

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crawfish (Procambarus clarkii), and crucian carp (Carassius auratus). It was

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observed that the method produces tenderness values that correlate (R2 = 0.910) well

23

with the sensory evaluation (p < 0.01). These results suggested that the breaking rate

24

of muscle fiber using the entropy weighted coefficient method can be quantified to

25

reflect meat tenderness, which is very suitable for cooked meat with low-fat, low

26

connective tissue, and meat which is hard to cut into specific shapes. The

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histochemical and textural properties were different for the different edible muscle

28

tissues. Tenderness had a high correlation with moisture, myofibrillar proteins and the

29

water holding capacity. Furthermore, the myofibrillar protein (about 70 g/100 g crude

30

protein) was the main determinant of tenderness. This study suggested that it might be

31

possible to develop a more widely applicable method for evaluating the cooked

32

texture of crustaceans.

33

Keywords: Chinese mitten crab, Eriocheir sinensis, horse crab, Portunus

34

trituberculatus, manila clam, Ruditapes philippinarum, red swamp crawfish,

35

Procambarus clarkii, crucian carp, Carassius auratus, tenderness

36 2

37

1. Introduction

38

Texture is the most important factor influencing consumer satisfaction for crab

39

meat palatability. The texture of crab meat is also an important part of the

40

comprehensive evaluation of crab quality. The outstanding texture characteristic of

41

aquatic products is their tenderness, but the water environment, circulation, and other

42

aspects will significantly affect the tenderness of crab meat, and there are also

43

differences in different muscle tissues. Tenderness is an important quality attribute

44

determined by the ease of chewing (Boleman et al., 1997). From the perspective of

45

consumer perception, it has been widely used in the evaluation of meat quality. The

46

tenderness of meat can be expressed as resistance to tooth pressure or how much force

47

it takes to bite through a piece of meat with molars. It can also be described by a large

48

number of descriptors, including mushy, crumbly, mealy, or elastic (KaiĆ and ŽGur,

49

2017; Marino et al., 2011).

50

Meat tenderness can be measured using instruments or humans (as sensory or

51

consumer groups). The instrumental methods for measuring shear force (SF),

52

including slice shear force (SSF) and Warner-Bratzler shear force (WBSF), have been

53

widely used in meat tenderness evaluation. WBSF is used to measure the strength

54

needed for whole muscle fiber shearing (AMSA, 2015). This method requires that the

55

sample be cut to a specified size so that the core perpendicular to the muscle fiber can

56

be cut completely. However, crab meat is hard to cut into specific shapes so it cannot

57

meet the sample requirements for instrumental measurements. Moreover, it has been

58

shown that shear force (both WBSF and SSF) does not properly reflect tenderness 3

59

differences among muscles (King et al., 2009; Rhee et al., 2004). Therefore, it is

60

inappropriate to use SF to compare tenderness differences among muscles (AMSA,

61

2015). The measurement of myofibril fragment lengths (MFL) after homogenization

62

is a method to microscopically predict meat tenderness. The myofibril sediment is

63

measured using phase contrast microscopy by counting the number of sarcomere

64

segments of the average length of each fibril using 100 fibrils (Moholisa et al., 2017;

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MØller et al., 1973; Pool and Klose, 1969). MØller et al. (1973) observed that

66

mechanical measurements of fragments reflected the physical properties of the muscle

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tissue and had a significant correlation with meat tenderness. Based on the micro

68

method, it might be possible to reflect tenderness or fragility differences among

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muscles using the rate of change of particle size over multiple time gradients using a

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Malvern Mastersizer. Since the method is not a one-time break to detect the breaking

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force, the breaking rate is monitored using different breaking times with the same

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breaking force, which is more consistent with multiple breaks during chewing, and

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might more objectively reflect the degree of fragility of different muscle fibers.

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For marine crabs species, snow crab (Chionoecetes opilio) (Hayashi et al., 1981),

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blue crab (Callinectes sapidus) (Çelik et al., 2004; Gökoðlu and Yerlikaya, 2003;

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Hernández-Robledo et al., 2016; Martínez et al., 2014), green crab (Carcinus maenus

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(Skonberg and Perkins, 2002), Carcinus mediterraneus (Cherif et al., 2008)), brown

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crab (Cancer pagurus) (Barrento et al., 2010), warty crab (Eriphia verrucosa) (Kaya

79

et al., 2009), mangrove crab (Scylla serrata) (Castilho et al., 2007), Atlantic spider

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crab (Maja brachydactyla) (Marques et al., 2010), swimming crab (Ovalipes

4

81

trimaculatus) (Dima, et al., 2012), stone crab (Platyxanthus patagonicus) (Dima et al.,

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2012), and mud crab (Scylla serrata) (Benjakul and Sutthipan, 2009), other than

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nutrition and flavor characteristics, previous studies have mainly focused on the

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muscle histomorphology, as well as changes in the physicochemical and sensory

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properties during thermal processing and frozen storage. However, few studies have

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focused on the texture properties of cooked crab meat, which could generally be

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measured using the water holding capacity and sensory evaluation as the standard.

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There is currently no quantitative method for determining tenderness of crab meat.

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This study used E. sinensis, which is quite popular among Chinese consumers

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due to its unique flavors and fine texture. The annual production of E. sinensis has

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increased sharply from 649,000 tonnes in 2011 to 812,000 tonnes in 2016 (China

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Fishery Bureau, 2012, 2017). The purpose of this study was to develop a method for

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evaluating the tenderness of cooked crab meat and investigate the correlation between

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tenderness and histochemistry of edible muscle tissues from E. sinensis, as well as

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identify the important histochemical compounds affecting the tenderness of different

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muscle tissues from E. sinensis.

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2. Materials and methods

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2.1 Materials

99

Perchloric

acid

(PCA),

NaCl,

CuSO4,

NaOH,

Tris

(hydroxymethyl)

100

aminomethane (Tris) and E-51 resin were obtained from Sinopharm Chemical

101

Reagent

102

N,N,N´,N´-tetramethylethylenediamine (TEMED), sodium dodecyl sulphate (SDS),

Co.

(Shanghai,

China).

Coomassie

blue

R-250,

bis-acrylamide,

5

103

and high and low molecular weight markers were bought from Bio-Rad Laboratories

104

(Hercules, CA, USA). Carnoy's solution was bought from Beijing Solarbio Science &

105

Technology Co., Ltd. (Beijing, China). HPLC-grade glutaraldehyde, 100% ethanol,

106

and acetone were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA).

107

All other chemicals were analytical grade from Ampel Lab Technologies Inc.

108

(Shanghai, China).

109

2.2 Muscle samples

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Twenty males (used for all analyses, 110 ± 4 g) and 6 females (only used for

111

biometric data analysis, 110 ± 4 g) E. sinensis were harvested in April, 2018, by a

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crab company (Chongming Island, Shanghai, China), and promptly tied individually

113

using cotton rope and transported alive to the laboratory within 2 h in a styrofoam box

114

with refrigeration (~4oC). The muscle tissues (including claw, abdomen and leg

115

muscles), gonads, and hepatopancreas as the edible parts were individually removed

116

and weighed (BT224S, Sartorius Lab Instruments GmbH & Co. KG, Goettingen,

117

Germany) in a 4-7oC chill storage. For each tissue, yield was calculated as follows:

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Yield = each edible part wet weight/body wet weight×100

(1)

119

Edible Contribution = each edible part weight/sun of edible parts weight×100

(2)

120 121

Each muscle was subsequently collected, mixed, vacuum packed and stored at -70oC for further analysis (a maximum of 4 wk).

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P. trituberculatus (male, n = 8, 165 ± 5 g), R. philippinarum (n = 60, 20 ± 1 g), P.

123

clarkia (n = 50, 27 ± 2 g), C. auratus (n = 10, 155 ± 5 g) were purchased at the

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Shanghai seafood market in April, 2018. Whole live samples were steamed at 100oC

6

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for 20 min, then various cooked meat of E. sinensis (from claws, legs, and abdomen),

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P. trituberculatus (from abdomen), R. philippinarum (from adductor), P. clarkii (from

127

abdomen), and C. auratus (from dorsal body) were manually separated using tweezers

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with the help of laboratory members and used for tenderness measurements.

129

2.3 Proximate composition analysis

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The moisture was determined using drying in the oven at 105oC until a constant

131

weight was achieved according to analytical method No. 950.46 (AOAC, 2000). The

132

crude protein was determined using the Kjeldahl method No. 920.153 (AOAC, 2000).

133

The conversion factor of total nitrogen to crude protein was 6.25. The fat was

134

determined using the Soxhlet extraction method No. 960.39 (AOAC, 2000). The ash

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was determined using ashing for 8 to 12 h at 550oC according to analytical method No.

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928.08 (AOAC, 2000).

137

2.4 Fractionation of nitrogenous constituents

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Proteins in raw crab muscles (including claws, abdominal and legs muscles)

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were classified separately according to solubility using the method of Hashimoto et al.

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(1979). All operations were done at 4oC. All muscle samples (2.5 g) were put into 10

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volumes of buffer A (0.05 M NaCl, 20 mM Tris-HCl, pH = 7.0), and samples were

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homogenized at 14,000 rpm for 1.5 min (HG-200, Hsiangtai Machinery Industry Co.,

143

Ltd., Taipei, Taiwan). Samples were stirred for 0.5 h using a mechanical stirrer (RT10,

144

IKA®-Werke GmbH & Co., KG, Staufen, Germany) at a speed of 300 rpm. The

145

extract was centrifuged using a refrigerated centrifuge for 10 min at 12,000×g (9,960

146

rpm in a JA-12 rotor, Avanti® J-26XP, Beckman Coulter, Inc., Palo Alto, CA, USA).

7

147

Centrifugation was done 3 times and all supernatants were combined. PCA was added

148

to the solution and the final concentration was 5% (w/v). The mixture was centrifuged

149

at 12,000×g for 5 min at 4oC. The precipitate was mainly the sarcoplasmic proteins

150

and the remaining supernatant was considered to be non-protein nitrogen, which

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included other nitrogen compounds, including amino acids and small peptides.

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The residue after extraction with buffer A was stirred with 10 volumes of buffer

153

B (0.5 M NaCl, 20 mM Tris-HCl, pH = 7.0) for 0.5 h, and centrifuged at 12,000×g for

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10 min. The extraction was done 3 times. The supernatants were combined into the

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myofibrillar protein fraction. Ten volumes of 0.1 M NaOH were added to the

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precipitate, and the mixture was stirred continuously for 2 h. The mixture was then

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centrifuged at 12,000×g for 10 min. The extraction was done 4 times. The 4

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supernatants were combined into the alkali-soluble protein fraction. The final

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precipitate was the stroma protein fraction (alkali-insoluble protein fraction).

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Total Kjeldahl nitrogen contents (AOAC, 2000) of the 5 fractions were obtained

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along with their protein patterns.

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2.5 SDS–polyacrylamide gel electrophoresis (SDS–PAGE)

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The protein profiles of raw crab muscles were determined using SDS-PAGE as

164

described by Laemmli (1970) with slight modifications. The protein samples were

165

dissolved in electrophoresis buffer solution (containing 2% SDS, 8 M carbamide, 2%

166

β-mercaptoethanol, 50 mM Tris-HCl, pH = 8.0) and heated at 100oC for 3 min. The

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samples (10 µL, contained ~10 µg of protein) were loaded on homemade 7.5%

168

separating gel and 5% stacking gel, and electrophoresis was carried out with a

8

169

constant current of 15 mA/gel. After gel electrophoresis, the gel was stained with

170

0.1% Coomassie brilliant blue R-250 in a mixed solution of 45% methanol and 10%

171

acetic acid for 30 min and destained with 45% methanol and 9% acetic acid for 2 h.

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Nine molecular weight markers, including myosin heavy chain from rabbit skeletal

173

muscle (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97.2 kDa), serum

174

albumin (66.4 kDa), ovalbumin (44.2 kDa), carbonic anhydrase (29 kDa), trypsin

175

inhibitor (20.1 kDa), lysozyme (14.3 kDa) and aprotinin (6.5 kDa), were used for

176

estimating protein sizes.

177

2.6 Histological analysis

178

Specimen preparation for light microscopy (LM) was done using the method of

179

Lee et al. (2012). Raw muscles were placed for 40 min in Carnoy's solution,

180

embedded in paraffin wax, sliced to a thickness of 4-6 µm using a microtome

181

(RM2235, Leica Microsystems Vertrieb GmbH, Wetzlar, Germany), and stained with

182

hematoxylin and eosin. Histological observations were done using an optical

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microscope (DMI3000, Leica Microsystems) using 100 and 400 × magnification.

184

Specimens were prepared for transmission electron microscope (TEM) using the

185

method of Zhen et al. (2018). Muscle tissues (~1 mm3 cube) were separated using a

186

sharp blade from raw muscles, then fixed in 2.5% glutaraldehyde for 3 h at 4oC,

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rinsed in 0.1 M sodium phosphate buffer, and then post-fixed in 1% osmium tetroxide

188

(OsO4) solution for 2 h at 4oC. After fixation, 0.1 M phosphate buffer was used to

189

wash the specimens 3 times for 20 min, the specimens were dehydrated for 15 min

190

each with a series of ethanol and acetone solutions (50, 70 and 90% ethanol, 90%

9

191

ethanol:90% acetone at a 1:1 ratio, 90% acetone, and 100% acetone each 3 times). The

192

samples were embedded in E-51 resin and sliced into ultra-thin sections (60 nm in

193

thickness). The samples were placed on copper mesh (200 mesh/inch) and stained

194

with 3% uranyl acetate and 3% lead citrate. The specimens were observed using a

195

TEM (CM-120, Philips Electronics Ltd., Eindhoven, The Netherlands) at 60 kV.

196

2.7 Low field nuclear magnetic resonance (LF-NMR) measurements

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LF-NMR was used to measure the distribution of water in muscle tissue, which

198

was done using a Niumag Benchtop Pulsed NMR Analyzer MesoMR23-060H-I

199

(Niumag Electric Corp., Shanghai, China) operating at a resonance frequency for

200

protons of 21 MHz. The measurements were done using the method reported by Shao

201

et al. (2016). The relaxation times using low-field 1H NMR were measured in 3.0 g

202

portions of fillet placed in sampling tubes (70 mm in diameter). Spin-spin relaxation

203

times (T2) were collected using a Carr-Purcell-Meiboom-Gill pulse sequence with the

204

echo times of 500 µs and 2,000 collected echoes at 25oC. T2b, T21 and T22 relax in the

205

range of 0-10, 30-100, and >200 ms, respectively. The scan was repeated 3 times.

206

2.8 Water holding capacity (WHC)

207

Centrifugal and cooking loss reflected the WHC (Li et al., 2017). The WHC was

208

measured using centrifuge drip and cooking loss. Approximately 3.0 g of raw crab

209

muscle was weighed in a dry, clean centrifuge tube. The crab muscle was then

210

centrifuged at 12,000×g at 4oC for 15 min, the liquid at the top of tubes was removed,

211

and the meat samples were weighed to determine the centrifuge drip. The following

212

equation was used:

10

213

Centrifuge Drip = 100×(1−A/B)

214

where A is the final weight of the sample, and B is the initial weight of the sample.

215

(3)

The cooking loss of crab meat was measured after steaming. The samples (3.00 g)

216

were placed in a 5 × 7 cm2 polyethylene bag (Jiangyin Honghao Packaging Materials

217

Co., Ltd., Jiangyin, Jiangsu, China), then steamed at 100oC for 3 min and immediately

218

cooled on ice. The meat samples were removed from the bag and placed in a 50 mL

219

centrifuge tube with a 0.25 mm diameter filter screen (purchased at the Xinxing

220

experimental equipment market in Haimen, Jiangsu, China) to allow additional drip

221

for 10 min at 4oC. The cooking loss equation was:

222

Cooking Loss = 100×(1−A/B)

223

where A is the weight after cooking, and B is the weight before cooking.

224

2.9 Instrumental tenderness measurement

(4)

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Meat samples (3 g) from cooked E. sinensis (including claws, abdomen, and

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legs), P. trituberculatus, R. philippinarum, P. clarkii, and C. auratus were finely

227

blade-minced and added to 5 volumes (v/w) of buffer A (0.05 M NaCl, 20 mM

228

Tris-HCl, pH = 7.0) in a 50 mL centrifuge tube, then homogenized for 30, 45, 60, 75,

229

and 90 s using the homogenizer at a speed of 8,000 rpm in the centrifuge tube. Each

230

homogenate was used for particle size analyses using a Malvern Mastersizer (MS2000,

231

Malvern Instruments Ltd., Malvern, UK).

232

With constant stirring, the samples were diluted to 1:500 with distilled water in

233

the sample cell at 20oC. The particle size was measured in the range of 0.02-2,000 µm.

234

The specific parameters (Gao et al., 2012) were set as follows: the refractive index of

11

235

default particle (glass: 1.520), the particle absorbance value (0.1), and the refractive

236

index of water (1.330). Each measurement was repeated 3 times. The data are

237

represented by characteristic parameters, including D[3,2], D[4,3], d(0.1), d(0.5), and

238

d(0.9). D[3,2] is the Sauter mean particle diameter, which refers to the average

239

diameter of the equivalent sphere with the same specific surface area as the actual

240

particle group. D[4,3] is the volumetric mean particle diameter, which represents the

241

equivalent sphere of the same volume as the actual particle group. d(0.1), d(0.5), and

242

d(0.9) are the volumetric diameters at which 10, 50, and 90% of the particles are

243

smaller (Liu et al., 2008).

244

After treatment with different homogenization times (30, 45, 60, 75, and 90 s),

245

the rate of the particle size changes of the homogenate were measured. The

246

corresponding weighted coefficient of each characteristic parameter was obtained

247

using the entropy weighted coefficient method (Jia and Zhao, 2010; Zhong, 2017).

248

The characteristic of the entropy weighted coefficient method is that the weighted

249

coefficient of each characteristic parameter is directly calculated using the rate of the

250

particle size changes with each characteristic parameter, and the subjective judgment

251

of the decision maker is not introduced. The instrument tenderness (total breaking rate)

252

equation was as follows:

253

Instrument Tenderness = aKD[3,2]+ bKD[4,3]+ cKd(0.1)+ dKd(0.5)+ eKd(0.9)

254

where a, b, c, d, and e are the corresponding weighted coefficient of Ki, Ki is the rate

255

of the particle size changes with each characteristic parameter.

256

(5)

Finally, the correlation of instrument tenderness value and sensory tenderness

12

257

value was measured to verify the accuracy and reliability of the instrument for

258

evaluating meat tenderness.

259

2.10 Sensory analysis

260

Sensory analysis was done on cooked meat from the 5 species. Sensory analysis

261

for tenderness was done using a 10-member trained descriptive attribute panel that

262

was screened, selected, and trained in accordance with the guidelines for the sensory

263

evaluation of meat of the American Meat Science Association (AMSA, 2015). The

264

panel consisted of 10 members aged 20-30, 3 males and 7 females from Shanghai

265

Ocean University. Sensory experiments were carried out in a normal sensory

266

laboratory, which complied with international standards for test rooms (ISO, 2014).

267

The test rooms are designed to do sensory evaluations using known and controlled

268

conditions with a minimum of distractions and to reduce the effects that psychological

269

factors and physical conditions can have on the panelists judgement. The testing area

270

is separate to reduce interference, such as from odour and noise. The colour of the

271

walls and furnishings of the testing area is light neutral grey, and the lighting is

272

uniform, free from strong shadows. Prior to sensory evaluation, the panelists had

273

thoroughly discussed 18 aquatic meats including fish, shrimp, shellfish and crab with

274

words to identify the extremes and middle of the rating scale. As training progressed,

275

the panelists were able to identify other points along the rating scale. The tenderness

276

was measured using a quantitative descriptive analysis (QDA) scale with a 9-point

277

descriptive scale (1 = Extremely Tough, 2 = Very Tough, 3 = Moderately Tough, 4 =

278

Slightly Tough, 5 = Neither Tender nor Tough, 6 = Lightly Tender, 7 = Moderately

13

279

Tender, 8 = Very Tender, 9 = Extremely Tender).

280

2.11 Statistical analysis

281

Results were shown as mean values ± standard deviation (SD). The one-way

282

analysis of variance (ANOVA) with Duncan's multiple range test was used to analyze

283

the data. The Statistical Package for the Social Sciences (SPSS for Windows: SPSS

284

Inc., Chicago, IL, USA) was used for statistical analysis and Pearson's method was

285

used for the correlation analysis. A p-value < 0.05 was considered statistically

286

significant. However, 0.01 was also used for some of the data to indicate the greater

287

significance of the differences. All analyses were repeated 3 times.

288

3. Results and Discussion

289

3.1 Proximate compositions

290

The average meat yield from E. sinensis was ~37%, in which muscle made the

291

greatest contribution, followed by the hepatopancreas, and gonads (Table 1). The

292

muscle contributions from E. sinensis were higher than those compared with other

293

marine crab species. For example, Barrento et al. (2010) reported that the muscle and

294

hepatopancreas of brown crabs were responsible for the majority of meat yield, with

295

muscle being a larger percentage in male crabs compared to females. The Atlantic

296

spider crab had an average meat yield of ~17%, in which the hepatopancreas had the

297

highest contribution, followed by muscle tissue and the gonads (Marques et al., 2010).

298

This study confirmed that males contributed a higher proportion of muscle from claws

299

than females of the same size. This species shows sexual dimorphism, displayed in

300

the larger claws of males compared with females. 14

301

Based on wet weight, the proximate compositions among all the tissues from E.

302

sinensis show that claw muscle had the lowest moisture and the highest crude protein

303

and ash (Table 2). On the other hand, the muscle from the abdomen had the highest

304

moisture and fat. The total fat in the three tissues was far below 5%, a value generally

305

considered to characterize a low-fat food. All muscle tissues had crude protein as the

306

major component. Based on dry weight, the abdomen muscle had the highest crude

307

protein, followed by claw muscle and leg muscle tissues. The abdomen muscle also

308

had the highest fat compared to the claw and leg muscles. The differences of ash were

309

not significant among the different muscles.

310

These results are consistent with muscle being the structural tissue with the

311

highest crude protein and poor fat. Previous studies, such as brown crab (Barrento, et

312

al., 2010) and warty crab (Kaya et al., 2009), are also consistent with these results.

313

For example, the claw muscle of brown crab from both sexes had 74.6-77.8%

314

moisture, 16.4-20.5% crude protein, 0.2-0.4% fat, and 1.9-2.2% ash (Barrento, et al.,

315

2010). For warty crab meat, the crude protein and crude lipid were 19.7 and 0.66

316

g/100 g wet weight (Kaya et al., 2009). The texture of muscle tissue is mainly

317

attributed to macromolecular substances. Because crude protein is the major

318

component of muscle, it has a leading role in the texture of crab meat.

319

3.2 Distribution of nitrogenous constituents

320

The various tissues of E. sinensis had different nitrogen distributions (Table 3)

321

and protein patterns (Fig. 1). The non-protein nitrogen-containing compounds were

322

mainly related to taste compounds, including amino acids, nucleotides, and peptides.

15

323

The sarcoplasmic fraction extracted from muscle tissue is made up of enzymes,

324

principally glycolytic enzymes, but other enzymes are present as well, including

325

enzymes related to the pentose shunt and auxiliary enzymes such as creatine kinase

326

and AMP deaminase (Strasburg et al., 2017). The non-protein nitrogen had a wet

327

weight of 0.17-0.25 g/100 g, and the sarcoplasmic fraction had a wet weight of

328

3.2-3.9 g/100 g (Table 3). Fig. 1 shows that proteins with a low molecular weight

329

(below 97 kDa) were present in the sarcoplasmic fractions of all tissues tested.

330

The myofibrillar proteins from invertebrate muscles have extraordinarily large,

331

thick filaments mainly composed of paramyosin (PM) forming a core and myosin as a

332

surface protein. Myosin and actin (AC) are the main participants in muscle

333

contraction (Shelud’Ko et al., 1999). The myofibrillar fraction was dominant in three

334

muscle tissues with significant differences of content. The myofibrillar fraction from

335

three muscle tissues showed similar SDS-PAGE patterns, showing myosin heavy

336

chain (MHC) and AC as the major proteins. Myosin is the most abundant protein in

337

the myofibril and provides an important contribution to the structure and tensile

338

strength of meat (Marino et al., 2013). PM, which has a molecular weight of 100 kDa,

339

was also observed, and the PM in the claw muscle was higher than the content in

340

either the abdomen or leg muscle. Based on the functions of these muscles, PM could

341

contribute to the strength of the corresponding muscle tissue. Moreover, thermally

342

induced gelation of PM from scallop smooth adductor muscle was very firm and

343

brittle. The PM thermal gel was made at temperatures above 30°C using a two-step

344

increase in elastic (G′) and viscous (G′′) moduli, these values were higher than in gels

16

345

produced from actomyosin at a high temperature (Fukuda et al., 2006).

346

Alkali soluble components arise from polymerized myofibrillar proteins

347

(Karnjanapratum et al., 2013). Proteins with a molecular weight of ~35, 45, 100, and

348

200 kDa were observed in the alkali-soluble fraction, corresponding to tropomyosin

349

(TM), AC, PM, and MHC, respectively. Insoluble components were regarded as

350

stroma proteins. The connective tissues encasing individual muscle fibers

351

(endomysium), muscle fiber bundles (perimysium), and muscle (epimysium)

352

contributed to the “background toughness” of meat (Xiong, 2018). It should be noted

353

that the stroma proteins in the muscle tissues of E. sinensis was low. Collagen is a

354

major component of stroma proteins of connective tissue (Xiong, 2018). Mizuta et al.

355

(1994) reported that the muscles of prawn and shrimp, which had relatively high

356

collagen, tended to be firm, whereas the muscles in crabs were generally less firm and

357

the collagen was typically lower. High molecular weight (above 200 kDa) proteins

358

were still observed in the stroma fraction (Fig. 1).

359

3.3 Observations of muscle fiber structure using LM and TEM

360

To better understand the contribution of muscle fiber and connective tissue to

361

meat texture, it is important to clarify the distribution of these tissues. The transverse

362

and longitudinal sections of the raw muscle tissues were observed (Fig. 2A) using an

363

optical microscope. Muscle fiber and connective tissue were stained bright red and

364

light red, respectively (Fig. 2A). Connective tissue was relatively poor in areas

365

primarily consisting of muscle tissue. A similar pattern was observed for the muscles

366

from prawn and shrimp, which showed a dense distribution of collagen fibers, while

17

367

the muscles from crabs had a sparse distribution as reported by Mizuta et al. (1994).

368

Those findings were consistent with the very low muscle stroma protein observed in

369

the nitrogen distribution analyses. The muscle bundle diameter of muscle from the

370

abdomen was the largest, followed by the claw and leg (Fig. 2A).

371

Further observations of the ultrastructure of the raw muscles using longitudinal

372

sections allowed for the determination of striated muscles formed by overlap of thin

373

and thick filaments (Fig. 2B). Franzini (1970) observed that the length of thin and

374

thick filaments was highly variable within a single crab’s fiber, therefore, a

375

comparison made on the basis of a few random measurements would not valid.

376

Sarcomere length is a measure of the state of postmortem raw muscle shortening

377

(Locker and Hagyard, 1963), however, in this study, the sarcomere changes of raw

378

muscle in the post-mortem period were not investigated.

379

In transverse sections, the lattice distribution formed by thick microfilaments was

380

visible, with thin filaments surrounding the hexagonally arranged thick filaments (Fig.

381

2B). The average diameters of thick microfilaments of the claw, abdomen and leg

382

were 21 ± 2, 23 ± 3, and 19 ± 2 nm (p < 0.05), respectively. The thick microfilaments

383

were rarely observed in a regular pattern in the three E. sinensis. On the other hand,

384

Squire et al. (1981) reported the thick microfilaments of vertebrate skeletal muscle

385

could form a regular lattice arrangement with 6 thick microfilaments arranged

386

equidistantly around each thick microfilament. Additionally, in other studies, the

387

reseachers observed that a more or less hexagonally shaped thick filament lattice was

388

invariably present in ultrathin transverse sections of marine crabs (Cohen and Hess,

18

389

1967; Franzini, 1970; Fourtner and Sherman, 1972; Hoyle, 1978). It was speculated

390

that this was closely related to the maturity of the crab and the tenderness of the

391

muscle. As the crab matures, the lattice regularity of the thick microfilaments

392

increased, and the tenderness of the muscle decreased. In future research, the

393

arrangement of thick microfilaments in cooked muscle should be studied.

394

3.4 Water distributions and WHC

395

The muscle tissue water was split into three segments for more detailed analyses.

396

Fig. 3 shows the water distributions with NMR, and WHC of the raw samples. It was

397

observed that the entrapped water was the highest, followed by bound water and free

398

water. Amongst all the raw muscles, muscle from the abdomen had the highest free

399

water, which could contribute to the higher moisture. The WHC of meat can be

400

defined as its ability to retain water and this has a direct influence on its tenderness

401

and yield, thereby influencing its economic value (Guichard, 2006). Centrifuge drip

402

and cooking loss are the primary indicators of WHC of raw and cooked meat,

403

respectively (Silva et al., 2015). The highest centrifuge drip and cooking loss were

404

observed in the abdomen muscle. The majority of the water that was affected by the

405

process of converting muscle to meat was the entrapped water. Bertram et al. (2002)

406

reported that more mobile myowater within the meat (extra-myofibrillar water) was to

407

be considered as the source of the potential drip from the meat. From the indices of

408

centrifuge drip and cooking loss, the claw muscle showed the highest WHC compared

409

to the abdomen and leg muscle.

410

3.5 Instrumental measurement and sensory evaluation of meat tenderness

19

411

Fig. 4 shows the distribution of particle size with different homogenization times.

412

The curve shifted to the left with the increasing of homogenization times, and the

413

specificity of the curve shape of different aquatic animals is shown. To quantify

414

tenderness, the variation of particle size with different characterization parameters is

415

shown in Fig. 5. The absolute value of the linear slope equation represents the

416

breaking rate of meat fiber with each characteristic parameter. The entropy weighted

417

coefficient method was used to calculate the corresponding weighted coefficient of

418

the breaking rate with each characteristic parameter and to obtain the instrument

419

tenderness value (Table 4). The weighted coefficients of D[3,2], D[4,3], d(0.1), d(0.5),

420

and d(0.9) were 27.9, 9.3, 40.3, 18.0, and 4.4%, respectively. The corresponding

421

instrumental tenderness values were ranked as: ES-claw > ES-leg > ES-abdomen >

422

CA-dorsal > PC-abdomen > PT-abdomen > RP-adductor. These results showed that

423

the breaking rate of the 3 edible muscles of E. sinensis was higher than that of other

424

species with the same homogenization speed, and the relationship between the

425

breaking rate of the muscles of E. sinensis was: claw meat > leg meat > abdomen

426

meat. In other words, crab meat was more tender than other species, and the claw

427

meat was the most tender, followed by leg meat. The tenderness correlation (R2 =

428

0.910) between the instrumental measurement and sensory evaluation was highly

429

significant (p < 0.01) (Fig. 6). Therefore, the breaking rate using the entropy weighted

430

coefficient method analysis could be quantified to reflect the tenderness of different

431

muscle tissues. When the same mechanical homogenization was used to shear the

432

meat, the particle size of the muscle fiber became smaller as the breaking times

20

433

increased. Moreover, for meat of different tenderness, the rate of reduction rate of

434

muscle fiber particle size was different, and the meat was more tender, and the

435

breaking rate was larger. The raw material properties were the basis to establish the

436

method of instrumental tenderness measurement. Therefore, this micro method is very

437

suitable for cooked meat with low-fat, low connective tissue, and meat which is hard

438

to cut into specific shapes compared with the shear force method.

439

3.6 Correlation analysis

440

The correlation analysis between various histochemical and textural indices of E.

441

sinensis are shown in Table 5. There was a positive correlation between moisture and

442

water loss, and a negative correlation between moisture and tenderness. Moreover,

443

water loss was negatively correlated with tenderness, which meant that the higher the

444

moisture, the weaker the WHC and less tender the cooked meat. A significant

445

relationship was observed between free water and the centrifuge drip (r = -0.785) and

446

the cooking loss (r = -0.842). The main constituent of fresh meat, water, was mainly

447

held within the highly organized structures of myofibrillar proteins. (Bertram et al.,

448

2006) A high amount of intra-myofibrillar water and a low amount of

449

extra-myofibrillar water might be associated with more tender meat (Pearce et al.,

450

2011). The distribution and mobility of intra-myofibrillar water were highly

451

associated with the structural features of the myofibrillar protein network (Wu et al.,

452

2006). The increased WHC would be consistent with higher protein-water interaction

453

and/or smaller interstitial water domains (Sánchez-González et al., 2008).

454

Myofibrillar protein is soluble in salt solutions, and is responsible for much of the

21

455

functional characteristics of fresh and processed meat (Xiong, 2018). These results

456

showed that myofibrillar protein was negatively correlated with water loss and

457

positively correlated with tenderness, which meant that a high myofibrillar protein

458

was associated with an improved WHC and tenderness of cooked meat. A significant

459

relationship was observed between the diameter of the thick microfilaments and the

460

entrapped water (r = 0.809). The change in the intra-myofibrillar myowater

461

characteristics was consistent with the increase in protein content/density of the

462

muscles (Xiong, 2018). However, no significant correlation was observed between the

463

diameter of the thick microfilaments and meat texture. Based upon the ultrastructure,

464

the irregular arrangement of the thick microfilaments might be closely related to the

465

high tenderness of E. sinensis meat. Little research has been done on the dynamic

466

changes of molecular information about functional groups, bonding types, molecular

467

conformations, and water distribution. Therefore, a subsequent study of the

468

protein-protein and water-protein interactions during the cooking would be beneficial.

469

4. Conclusion

470

The characteristics of food raw materials were shown using the histochemical and

471

textural characteristics of different muscles of E. sinensis. Based on histochemical

472

properties, it was observed that the breaking rate of muscle fiber after entropy

473

weighted coefficient method analysis could be quantified to reflect the meat

474

tenderness of cooked crab, which was very suitable for meat with low-fat, low

475

connective tissue, and cooked meat which is hard to cut into specific shapes.

476

Tenderness had a high correlation with moisture, myofibrillar proteins and the water 22

477

holding capacity. Ultrastructural and physicochemical properties could be the focus of

478

a follow-up study which would track the textural change of crab meat, analyze

479

physiological and biochemical changes of the myofibrillar protein with cooking the

480

reducing tenderness, and further identify proteins indicators of meat tenderness.

481

Acknowledgements

482

Authors acknowledge Professor Peng Yu at the Fudan University (Shanghai,

483

China) for the help with the TEM. This study was supported by grants from the 2018

484

National Key R&D Program of China (Grant No.2018YFD0901006), the 2018

485

National Natural Science Foundation of China (Grant No.31471608), and the 2018

486

Industrial System Project of Chinese Mitten Crab of Shanghai Municipal Agriculture

487

Commission (Grant No.D-8004-16-0179).

488 489

Conflict of Interest

490

The authors declare no competing financial interest.

491 492

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30

647

Table 1 The biometric data of edible tissues from E. sinensis Group

Yield

EC

648 649 650

Claw

Composition (g/100 g wet weight) Abdomen Leg Gonad

Hepatopancreas

Male

10±1 a

9±1 a

7±1 b

2.5±0.3 c

8±1ab

Female

3±0.2 c

9.9±1.2 a

8±1 ab

9.1±1.4 a

6.9±0.3 b

Male

26±1 a

25±3 ab

19±1 c

6.8±0.5 d

23±1 b

Female

8±0.1 c

26±2 a

23±3 a

24±2 a

19±2 b

Data are mean ± standard deviation (n = 6). Different lowercase letters in the same row indicate significant difference (p < 0.05). EC: Edible contribution.

31

651

Table 2 Proximate compositions of claw, abdomen, and leg muscles from E. sinensis Tissue Claw Abdomen Leg

652 653 654

Moisture 78±1 c

Composition (%) Crude Protein Fat

Ash

19±0.1 a

0.17±0.01 ab

2.5±0.3 a

(88±2) AB

(0.8±0.1) B

(11±1) A

81.7±0.5 a

16.5±0.1 b

0.2±0.1 a

1.8±0.1 b

80±0.1 b

(90±2) A 17±0.4 b

(1.3±0.3) A 0.13±0.01 b

(10±1) A 1.9±0.1 b

(85±2) B

(0.7±0.3) B

(9.6±0.4) A

Data are mean ± standard deviation (n = 3). Values in the parenthesis represent the content based on dry weight. Different lowercase or uppercase letters in the same column indicate significant difference (p < 0.05). ES: E. sinensis.

32

655

Table 3 Nitrogen distribution in claw, abdomen and, leg muscles from E. sinensis Nitrogen distribution (g/100 g wet weight) Tissue

Non-protein nitrogen

Sarcoplasmic

Myofibrillar

Alkali

Stroma

Claw

0.19±0.03 ab

3.24±0.03 c

14±0.3 a

1.6±0.2 a

0.16±0.01 b

(1.0±0.2) B

(17±0.2) C

(73±2) A

(8.3±1.2) A

(0.81±0.04) B

0.17±0.01 b

3.9±0.1 a

11±0.1 c

1.1±0.1 b

0.22±0.02 a

(1.04±0.03) B

(23±0.4) A

(68±1) B

(6.6±0.4) B

(1.3±0.1) A

0.25±0.05 a

3.7±0.1 b

12±0.1 b

0.66±0.04 c

0.19±0.03 a

(1.5±0.3) A

(22±1) B

(71±1) A

(4.0±0.2) C

(1.2±0.2) A

Abdomen Leg

656 657 658

Data are mean ± standard deviation (n = 3). Values in the parenthesis represent the content based on total protein. Different lowercase or uppercase letters in the same column indicate significant difference (p < 0.05).

659

33

660 661 662

Table 4 The absolute value (µm/s) of the linear slope equation and the corresponding weighted coefficients (%) with different characteristic parameters, and instrument tenderness value (µm/s) of different aquatic animals Tissue

663 664

D[3,2]

D[4,3]

d(0.1)

d(0.5)

d(0.9)

Tenderness

ES-claw

0.72±0.03a

2.2±0.1a

0.66±0.03a

2.1±0.1a

3.7±0.3a

1.21±0.05a

ES-abdomen

0.41±0.02c

1.5±0.1b

0.35±0.01c

1.44±0.03b

2.9±0.2b

0.79±0.03c

ES-leg

0.60±0.01b

1.4±0.1b

0.46±0.01b

1.50±0.04b

2.4±0.2c

0.87±0.02b

PT-abdomen

0.21±0.004e

0.9±0.1d

0.20±0.01d

0.78±0.05d

1.7±0.2d

0.44±0.03e

RP-adductor

0.03±0.01f

0.5±0.1e

0.01±0.002g

0.20±0.03e

2.1±0.6cd

0.19±0.04f

PC-abdomen

0.25±0.01d

1.07±0.05c

0.17±0.002e

0.80±0.05d

2.4±0.1c

0.48±0.01e

CA-dorsal

0.24±0.01d

1.5±0.1b

0.09±0.005f

1.1±0.1c

3.2±0.2ab

0.58±0.03d

Coefficient

27.9%

9.3%

40.3%

18.0%

4.4%

Data are mean ± standard deviation (n = 3). Different lowercase letters in the same column indicate significant difference (p < 0.05).

665

34

666

Table 5 The correlation analysis between various histochemical and textural indices

667

of E. sinensis.

668 669 670 671 672

Indices

Moisture

EW

FW

MF

DTF

CD

CL

IT

Moisture

1

EW

-0.177

1

FW

0.673*

-0.228

1

MF

-0.933**

0.211

-0.597

1

DTF

-0.214

0.809**

0.046

0.306

1

CD

0.907**

-0.097

0.785*

-0.921**

-0.03

1

CL

0.690*

0.069

0.842**

-0.748*

0.211

0.904**

1

IT

-0.934**

0.314

-0.533

0.970**

0.439

-0.856**

-0.615

1

ST

-0.706*

0.002

-0.658

0.725*

0.087

-0.803**

-0.799**

0.677*

ST

1

EW: Entrapped water, FW: Free water, MF: Myofibrillar protein, DTF: Diameters of thick microfilaments, CD: Centrifuge drip, CL: Cooking loss, IT: Instrumental tenderness, ST: Sensory tenderness, * Significant at the 5% level, ** Significant at the 1% level.

35

673

Figure captions

674

Fig. 1. SDS–PAGE patterns of different fractions of claw, abdomen, and leg muscles

675

from E. sinensis. W, S, M, A, T denotes whole portion, sarcoplasmic, myofibrillar,

676

alkali-soluble, and stroma fractions, respectively.

677

Fig. 2. Light microscopical microscopy (A) and transmission electron microscopy (B)

678

of the claw, abdomen, and leg muscles from E. sinensis. MB: muscle bundle.

679

Fig. 3. Water distributions, centrifuge drip, and cooking loss of the claw, abdomen,

680

and leg muscles from E. sinensis. BW, EW, FW, CD and CL denotes bound water,

681

entrapped water, free water, centrifuge drip, and cooking loss, respectively. Different

682

letters indicate significant differences (p < 0.05) between muscles.

683

Fig. 4. The variation of particle size with different characterization parameters.

684

Fig. 5. The distribution of particle size of different aquatic animals with different

685

homogenization times. ES, PT, RP, PC, and CA denoted E. sinensis, P. trituberculatus,

686

R. philippinarum, P. clarkii, C. auratus, respectively. Different letters indicate

687

significant differences (p < 0.05) between times.

688

Fig. 6. The correlation analysis between instrumental measurement and sensory

689

evaluation of 7 muscle tissues from different aquatic animals. Different letters

690

indicate significant differences (p < 0.05) between aquatic animals.

691

36

692 693 694

Fig. 1.

695

37

696 697 698 699

Fig. 2.

38

80 BW EW FW CD CL

Content ( g/100g wet weight )

a a

a

75

70 30

a

25 20

b

15 c

10 5

b

a b

a

a

a a

0 Claw

Abdomen

b b

Leg

700 701 702

Fig. 3.

703

39

704 705 706

Fig. 4.

707

40

D[3,2]

160

b

a

100

b

a

80

a

60

d

c

a

b

b

bc

b

c

c d d

c c

c d

d

a b

40 20

a

500

c

b

120

Partical Size (µ m)

b

Partical Size (µ m)

140

D[4,3]

600

a

c

c

300

200

a

c

c

c

30

45

60

75

90

b b

c b

30

b

45

d d

c c

c c

c c

60

75

90

d(0.5)

500

a

a 100

400

b

a 80

c d

b a b

60

e

c c

d

a b b

a

40

a

20

b

e

d b c

a

b

c c

30

45

60

e

c d

d e

c

Partical Size (µ m)

Partical Size (µ m)

c

d d

Time (s)

d(0.1)

120

c

b a a

300

c b b

b b

c d c c

a 200

a a 100

c

d d

b

c

d

c

b

bc

c

d

c c

c c

c c

60

75

90

b a

b

30

45

c 0

0 75

90

Time (s)

Time (s)

d(0.9)

1200

a

1000

b Partical Size (µ m)

c cd

d

b

Time (s)

a

800

a a a a

b b b

b b b

400

c

0

0

600

c

b b

b

a a

100

b

c b

a

a

c

a

b

a 400

a b

c c bc b

bc cd d c c

c

d

d e d

c

c

c

c

c c

60

75

90

200

ES-claw ES-abdomen ES-leg PT-abdomen RP-adductor PC-abdomen CA-dorsal

0 30

45

Time (s)

708 709

Fig. 5.

710

41

711 712 713

Fig. 6.

42

Highlights The breaking rate of muscle fiber based on particle size analysis can be quantified to reflect the tenderness of different muscle tissues from Eriocheir sinensis. This method is very suitable for cooked meat with low-fat, low connective tissue, and meat which is hard to cut into specific shapes. Higher myofibrillar protein suggested a stronger water holding capacity, and more tender meat.