14 2013, Vol.34, No.13 食品科学 ※基础研究

Effect of High Pressure Processing on Thermal Characteristics of Perimysium and

Endomysium Collagen from Beef semitendinosus Muscle

CHANG Hai-jun1，NIU Xiao-ying1

，TANG Cui1，WANG Qiang2

(1. Key Laboratory of Catalysis Science and Technology of Chongqing Education Commission, College of Environmental and

Biological Engineering, Chongqing Technology and Business University, Chongqing 400067, China；2. Department of Biological

and Chemical Engineering, Institute of Food Safety and Nutrition, Chongqing University of Education, Chongqing 400067, China)

Abstract：The main objective of this study was to investigate the effect of high pressure processing (HPP) (with varying

pressures and treatment time) on thermal characteristics of perimysium and endomysium collagen from beef. Muscle

samples were subjected to high pressure treatment varying from 200 to 600 MPa for 10 min and 20 min, respectively. The

changes of thermal shrinkage temperatures (To: onset temperature; Tp: peak temperature; Te: end temperature) of perimysium

and endomysium collagen of beef semitendinosus muscle following HPP treatment were analyzed by differential scanning

calorimeter (DSC). The results indicated that the effect of high pressure on thermal shrinkage temperatures of endomysium

collagen were more significant than that on perimysium collagen, especially when pressure was higher than 500 MPa and

held for longer than 20 min. At pressure above 500 MPa, longer treatment time could lead to higher shrinkage temperatures

of endomysium collagen (mainly presented in Tp). Pressures between 500 MPa and 600 MPa had a critical effect on the

thermal shrinkage temperatures of perimysium and endomysium collagen treated for 10 min and 20 min, respectively. The

changes in thermal shrinkage temperatures of perimysial and endomysial collagen of pressure processed beef muscle were

attributed to the pressure-induced changes of thermal characteristics (thermal stability) of connective tissue collagen.

Key words：beef semitendinosus muscle；perimysium and endomysium collagen；thermal shrinkage temperatures；high

pressure processing (HPP)；differential scanning calorimetry (DSC)

超高压处理对牛半腱肌肌束膜和肌内膜胶原蛋白热力学特性影响

常海军1，牛晓影1，唐 翠1，王 强2

(1.重庆工商大学环境与生物工程学院，重庆高校催化理论与应用技术市级重点实验室，重庆 400067；

2.重庆第二师范学院食品安全与营养研究所，生物与化学工程系，重庆 400067)

摘  要：探讨超高压处理(不同压力和保压时间)对牛半腱肌肌束膜和肌内膜胶原蛋白热力学特性影响。牛半腱肌

肉经200～600MPa压力分别处理10min和20min，用差示扫描量热法研究肌束膜和肌内膜胶原蛋白热力特性(起

始、最高和最终热收缩温度)在超高压处理过程中的变化。结果表明：超高压处理对牛半腱肌肌内膜胶原蛋白热

收缩温度的影响较肌束膜胶原蛋白显著，特别对于较高压力(500MPa及其以上)和较长保压时间(20min)的处理。

当压力超过500MPa时，保压时间越长，肌内膜胶原蛋白热收缩温度的增加越大(主要表现为最高热收缩温度)。

500～600MPa的压力处理是影响肌束膜和肌内膜胶原蛋白热收缩温度的关键处理压力。高压诱导的结缔组织胶

原蛋白热力特性(热稳定性)的变化是高压处理过程中牛肉肌束膜和肌内膜胶原蛋白热收缩温度产生差异的主要

原因。

关键词：牛半腱肌肉；肌束膜和肌内膜胶原蛋白；热收缩温度；超高压处理；差示扫描量热

中图分类号：TS251.1 文献标志码：A 文章编号：1002-6630(2013)13-0014-05

doi:10.7506/spkx1002-6630-201313004

收稿日期：2012-04-14

基金项目：国家自然科学基金项目(31101313)；重庆市教委科学技术研究项目(KJ110714；KJ121504)；

重庆市基础与前沿研究计划项目(cstc2013jcyjA80017)；重庆高校创新团队建设计划项目(KJTD201020)

作者简介：常海军(1980—)，男，副教授，博士，研究方向为畜产品加工理论与技术。E-mail：changhj909@163.com

※基础研究 食品科学 2013, Vol.34, No.13 15

Meat quality characteristics and textural properties both

have a crucial influence on the market success of meats and

meat products. The quality of meats is determined by their

sensory attributes, chemical composition, physical properties

and so on[1-3]. The texture of meat is one of the most important

quality properties, which has been studied for many years in

different aspects. High pressure processing (HPP) is one of

the emerging food processing high technology, which is non-

thermal, and consists of submitting the foods to pressures

above 100 MPa[4]. High pressure can modify the structure and

function of meat proteins and those changes affect the textural

and physiochemical properties of the muscle[5].

Changes of meat tenderness and texture during

pressure processing are partly resulted from the changes

of collagen characteristics, including collagen contents,

solubility and thermal stability (thermal characteristics)[6].

Meat collagen characteristics have been analyzed to obtain

information on meat tenderness, especially for collagen

contents and solubility. In addition to, the thermal stability

of connective tissue (perimysium and endomysium collagen)

has been analyzed by measuring the onset (To) and peak (Tp)

temperatures and enthalpy (∆H) of thermal shrinkage when

the role of intramuscular connective tissue (IMCT) in meat

tenderness has been studied[7].

Thus, the objective of this study was to investigate the

effects of high pressure processing (with different pressures

and maintain time) on thermal characteristics of perimysium

and endomysium collagen from beef semitendinosus muscle.

1 Materials and Methods

1.1 Materials

Beef semitendinosus samples were collected from 4

Chinese yellow bull (Nanyang × Simmental crossbreed)

(live weight: (500 ± 30) kg; age: 24－30 months) carcasses

slaughtered humanely in a commercial meat processing

company (Lüqi Meat Co. Ltd., Henan, China) by the Halal

method within 48 h postmortem, during which the carcasses

were hung by Achilles tendon in a 4 ℃ chiller (The cooling

process for meat ageing after slaughtering is a necessary

process). The visible subcutaneous fat and epimysial

connective tissue were trimmed off and sliced into 2.54 cm

thick cubes, perpendicular to the direction of the fiber. The

samples were prepared in triplicate.

1.2 Apparatus

HPP equipment (UHPF-750MPa, Kefa, Baotou, China);

MC-DSC (Multi cell differential scanning calometer, TA

instrument, USA); High-speed freezing centrifuge (Beckman

Allegra 64R, Beckman-Coulter Company, USA); Freeze-

drying system (Heto Power Dry LL3000, Thermo Scientific,

USA); Waring-basic blender (Ultra-Turrax T25, IKA-

WERKE, Germany); Drying oven (GZX-9076 MBE, Boxun,

Shanghai, China); Water bath (HH-42, Guohua, Changzhou,

China); Digital needle-tipped thermometer (HI145, Hanna

Instruments, Italy); pH meter (Thermo scientific, England);

Electronic Balance (AUY120, Shimadzu, Japan).

1.3 High pressure processing (HPP)

Beef semitendinosus muscle steaks (2.5 cm × 5.0 cm ×

5.0 cm; weight: (100 ± 5) kg) were vacuum packed with

polyethylene membrane layer (Beijing Huadun Xuehua

Plastic Group Co. Ltd., China) to prevent contamination

from the high-pressure transmission fluid (water). Raw

meat (untreated) kept at about 20 ℃ was used as the control

samples. Beef steaks were subjected to high pressure in

a 2 L vessel from 200 to 600 MPa for 10 min and 20 min

respectively at room temperature (20 ℃). The pressure level

and time of pressurization were controlled by a computer

program (BTNMC for HPP Control 1.0). Pressure holding

time reported in present study does not include pressure

come-up or release times. The pressure come-up rate was

350 MPa/min and pressures were released instantaneously.

Six muscle steaks for each pressure processing. After

processing, the surface water of muscle steaks were drained

by the absorbent paper, the steaks were stored under 4 ℃ for

index analysis after secondary vacuum packaging.

1.4 Perimysial and endomysial collagen preparation

The perimysial and endomysial portions of raw and high

pressure processed meat samples were prepared and extracted

according to the procedures of Light and Champion[8] modified

by Li Chunbao et al.[9]. Briefly, each meat sample (30 g wet

weight) was cut into 1 cm cubes and homogenized in ice-cold

50 mmol/L CaCl2 for 30 s at full speed (about 5000 r/min)

in a Waring Blendor. The homogenate was filtered through

a layer of nylon net (1 mm2 perforations) and the retentate

on the filter was rehomogenized in 50 mmol/L CaCl2 and re-

filtered. The process was repeated twice and then the filtrates,

containing endomysium, was collected. The retentate on

the filter was mainly the perimysium. The perimysium was

washed three times in 1 g/100 mL sodium dodecyl sulphate

(SDS) for 30 min at room temperature and SDS was removed

by dialysis at 4 ℃ against distilled water (24 h), 40%

methanol (24 h) and then distilled water (24 h).

16 2013, Vol.34, No.13 食品科学 ※基础研究

1.5 DSC analysis

DSC analysis of perimysium and endomysium were

conducted as described by Chang Haijun et al.[10] with slight

modifications. The purified perimysial and endomysial

portions were dried by freeze-drying, and then the thermal

shrinkage temperatures of perimysial and endomysial

collagen were measured using DSC (Multi cell differential

scanning calometer). The samples (10 mg) were accurately

weighed in aluminum pans and hermetically sealed. The

samples were heated from 20 to 100 ℃ at heating rate of

2 ℃/min. An empty sample pan was used as the reference.

Thermal shrinkage temperatures (To: onset temperature; Tp:

peak temperature; Te: end temperature) of perimysium and

endomysium collagen were estimated from the thermogram

using the software of Universal Analysis 2000 (TA

instrument).

1.6 Statistical analysis

All measurements in the study were done in triplicate;

the results reported here were the means of the three

replicates (expressed as the mean ± standard deviation).

Statistical analyses were carried out using Statistical

Package for the Social Sciences (SPSS) 16.0 (SPSS Inc.,

Chicago, IL). One-way analysis of variance (ANOVA) and

Duncan’s multiple-range test were carried out to determine

significant differences in perimysial and endomysial

portion contents, and thermal shrinkage temperatures (To,

Tp and Te) of perimysial and endomysial collagen between

pressure processed meat samples for 10 min and 20 min,

and the effects were considered significant at P＜0.05 (*)

and P＜0.01 (**).

2 Results and Analysis

2.1 Perimysial and endomysial portion contents

Perimysial and endomysial portion contents for beef

semitendinosus muscle during high pressure processing

are shown in Fig. 1 A and B respectively. Compared

with the control group, the perimysial portion content

for pressure treated meat (pressure maintain time were

10 min and 20 min) were decreased. There was a gradual

decrease in perimysial portion content with increasing in

pressure when the pressures maintain time was 10 min.

The endomysial portion content was showed the trend

of increased firstly and then decreased. The contents

of perimysial and endomysial portion from meat steak

after pressure processed were exhibited the significant

differences (P＜0.05 or P＜0.01) between pressure

processed for 10 min and 20 min during some treatments.

Perimysial and endomysial were the main components of

IMCT. Light et al.[11] have inferred that in muscle toughness,

the amount of perimysial collagen is much more important

than the amount of endomysial collagen. They also showed

that the variations of the amount of IMCT among muscles are

mainly because of the amount of perimysium, whereas, the

amount of endomysium remains almost constant. Therefore,

it seems essential to elucidate the mechanical properties of

the perimysium network in order to understand the role of

connective tissue in meat toughness.

*****

0123456

Control 200 300 400 500 600

Pressure/MPa

Perim

ysiu

m c

onte

nt/%

10 min 20 min

A

***

**

02468

10121416

Control 200 300 400 500 600Pressure/MPa

Endo

mys

ium

con

tent

/% 10 min 20 minB

*. Indicates the effects were considered significant at P＜0.05; **. Indicates

the effects were considered significant at P＜0.01, between pressure

processed meat samples for 10 min and 20 min. The same as follows.

Fig.1 Changes in perimysial (A) and endomysial (B) portion content

of intramuscular connective tissue from beef semitendinosus muscle

following HPP treatment

2.2 Thermal characteristics of perimysium collagen

Thermal shrinkage temperatures, including onset (To),

peak (Tp) and end (Te) temperature of perimysium collagen

were shown in Fig. 2 A, B and C respectively. As can be

seen from the figures, To and Tp of perimysium collagen

were manifested the same variation trends during the high

pressure processing. And the values of To and Tp for pressure

processed meat steaks for 20 min were higher than treated for

10 min excepted at the 600 MPa. There were fewer changes

in the Te of perimysium collagen for pressure processed beef

meat samples.

※基础研究 食品科学 2013, Vol.34, No.13 17

05

101520253035404550

Control 200 300 400 500 600Pressure/MPa

T o/

10 min 20 minA

0102030405060

Control 200 300 400 500 600Pressure/MPa

T p/

10 min 20 minB

0102030405060

Control 200 300 400 500 600Pressure/MPa

T e/

10 min 20 minC

Fig.2 Changes in thermal shrinkage temperatures of perimysium

collagen of beef semitendinosus muscle following HPP treatment

2.3 Thermal characteristics of endomysium collagen

The thermal characteristics of endomysium collagen

analyzed by DSC during high pressure processing are

depicted in Fig.3.

**

05

101520253035404550

Control 200 300 400 500 600Pressure/MPa

T o/

10 min 20 minA

* **

303234363840424446

Control 200 300 400 500 600Pressure/MPa

T p/

10 min 20 minB

*

0102030405060

Control 200 300 400 500 600Pressure/MPa

T e/

10 min 20 minC

Fig.3 Changes in thermal shrinkage temperatures of endomysium

collagen of beef semitendinosus muscle following HPP treatment

The effects of high pressure on thermal shrinkage

temperatures of endomysium collagen were more significant

than on perimysium collagen, especially for higher

pressure (≥500 MPa) and longer maintain time (20 min)

processing. When the pressure more than 500 MPa, the

longer pressure processed time (maintain time) can lead to

the higher shrinkage temperatures of endomysium collagen

(mainly presented in Tp) (P＜0.01), the peak temperature

of endomysium collagen from meat steak after pressure

processed for 20 min were higher than processed for 10 min

when the pressure up to 500 MPa (P＜0.05) and 600 MPa

(P＜0.01) respectively. This was probably resulted from the

weakening of the average stability of collagen because of the

pressure induced gelation and denaturation of endomysium

portion collagen.

According to Bailey et al.[12] reports, Tp of collagen

from mammals is around 65 ℃ but it is different for

different muscles and animal species. They reported that To is considered to describe the least stable collagen and

the Tp is a measure of the average stability of collagen.

In this study, perimysial and endomysial portions were

not purely the connective tissue collagen portions, this

was because that the mixed myofibrillar and connective

tissue proteins make it more difficult to homogenate and

separate perimysial and endomysial protein from the

mixed components perfectly. Although the DSC samples

were not pure collagen in this study, the thermal shrinkage

temperatures of collagen were similar to that reported on

whole meat. Nevertheless, minor difference also existed

because of the different muscles, animal species and

collagen kinds.

High hydrostatic pressurization is one of the new

techniques for tenderizing meat. The changes in the

ultrastructure of the pressurized muscle have been reported by

many workers[13-15]. The effects of high pressure processing on

meat tenderness (or toughness) resulted form the changes of

both “actomyosin toughness” and “background toughness”.

18 2013, Vol.34, No.13 食品科学 ※基础研究

Actomyosin toughness is the toughness attributed to the

myofibrillar protein, whilst the background toughness is the

toughness due to the presence of connective tissue collagen

and other stromal protein[16].

High hydrostatic had a significant effect on meat

histological structure and the texture of treated meat relies

on the gel network formed with the melted collagen,

denatured and aggregated myofibrillar proteins and

sarcoplasmic proteins. Pressure processing can lead to

the loss of the structural continuity of muscle due to the

rupture of I-filaments, loss of M-line protein and cleavage

of A-filaments[16], as well as the solubilization and gelation

of IMCT collagen (mainly comprising perimysial and

endomysial collagens). Based on those viewpoints, the

authors concluded that the changes in thermal shrinkage

temperatures of perimysial and endomysial collagen for

pressure processed beef muscle were attributed to the

pressure-induced changes in thermal characteristics (thermal

stability) of connective collagen.

2.4 Correlation analysis

Correlation coefficients among the traits of perimysium

and endomysium portion contents and thermal characteristics

(Tp) were listed in Table 1. The results showed that there

were no significant correlation between contents and thermal

shrinkage temperature, however, perimysium content were

correlated negatively (P＜0.01) with endomysium portion

content for pressure processed beef muscle (r =－0.526).

Table 1 Correlation between thermal characteristic changes of

perimysium and endomysium portion collagen following HPP treatment

(n=33)

Item PC EC P-Tp E-Tp

PC 1 －0.526** －0.003 0.216

EC 1 －0.326 0.262

P-Tp 1 0.030

E-Tp 1

Note: PC. Perimysial portion content; EC. Endomysial portion content; P-Tp.

Perimysium peak thermal shrinkage temperature; E-Tp. Endomysium peak

thermal shrinkage temperature; **. Correlation is significant at P＜0.01.

3 Conclusion

According to these results, the thermal stabilities

(expressed as thermal shrinkage temperatures) of perimysium

and endomysium collagen in beef semitendinosus muscle

changed during high pressure processing. Due to the pressure

exposure, perimysium and endomysium collageneous tissues

denatured and melted when the pressure up to 500 MPa,

and 500—600 MPa was critical treating pressure which

affects thermal shrinkage temperatures of perimysium and

endomysium collagen during processed for both 10 min and

20 min. Based on those viewpoints, it is concluded that the

changes in thermal shrinkage temperatures of perimysial

and endomysial collagen for pressure processed beef muscle

were attributed to the pressure-induced changes in thermal

characteristics (thermal stability) of connective tissue

collagen.

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