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High Pressure Research

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High-pressure effects on cooking loss and histological structure of beef muscle

Anjun Liu , Hu Zhan , Jie Zheng , Dongyue Liu & Peiqi Jia
a a a a a b

Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin, People's Republic of China
b

Tianjin HTSM Bioengineering Co., Ltd., Tianjin, People's Republic of China Available online: 03 Nov 2010

To cite this article: Anjun Liu, Hu Zhan, Jie Zheng, Dongyue Liu & Peiqi Jia (2010): High-pressure effects on cooking loss and histological structure of beef muscle, High Pressure Research, 30:4, 538-546 To link to this article: http://dx.doi.org/10.1080/08957959.2010.526940

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High-pressure effects on cooking loss and histological structure of beef muscle

Anjun Liua *, Hu Zhana , Jie Zhenga , Dongyue Liua and Peiqi Jiab
a Key

Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin, Peoples Republic of China; b Tianjin HTSM Bioengineering Co., Ltd., Tianjin, Peoples Republic of China
(Received 13 July 2010; nal version received 22 September 2010 )

In this study, we investigate the effects of high pressures (up to 600 MPa) applied at room temperature for 10 min on beef cooking loss and structure. The data on cooking loss, pH and protein solubility, as well as the electron microscopy, illustrate the changes in cooking loss and structure with high pressure processing (HPP). There is a signicant reduction in cooking loss of beef with HPP. When the beef sample is imposed upon by 300 or 400 MPa, the cooking loss reduction is about 12%. Further, the pH of beef is dramatically increased as the pressure increases, and the pH increases by about 5% when imposed upon by 500 MPa. When a high pressure was applied at room temperature, the structure of the beef tissue apparently changed. Muscle ber fragments gradually became slender and sarcomeres became lengthened. Our data indicated that high-pressure treatment on beef leads to stretching of the muscle ber and an increase in the water-holding capacity. Keywords: high pressure; cooking loss; protein solubility; structure; beef muscle

1.

Introduction

As a potential improvement in the standard of living, the quality of beef commands considerable attention. The water-holding capacity (WHC) of beef is an important value that evaluates the quality of meat, and concerns the ability of maintaining the intrinsic and additional water when the muscles are in the circumstance of external force. WHC is not only a factor that affects the meats color, avor, juiciness, tenderness and other qualities, but is also an important economic value because of its positive correlation with the production rate. In recent years, high-pressure processing (HPP), a food-processing method, has shown great potential in the food industry. HPP technology has become a focus in the meat product industry. The Australian scientist Macfarlane [1] rst reported that high pressures (up to 200 MPa) could increase the tenderness of beef. Other investigators began to study the effects of high pressures

*Corresponding author. Email: laj@tust.edu.cn

ISSN 0895-7959 print/ISSN 1477-2299 online 2010 Taylor & Francis DOI: 10.1080/08957959.2010.526940 http://www.informaworld.com

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on various characteristics of meat and made great achievements, concentrating on the effects of high pressures on tenderness, avor, color, gel and other aspects of meat. A recent study of the tenderness of raw meat shows that a massive decrease in the hardness of meat at 200 MPa and 70 C, which indicates an increase in protease activity [2]. Yet other studies indicate that high pressures caused a slightly hard texture [3]. Further, high pressure treatment may change the structure of protein and affects the quality of meat products. It has been proved that the denaturation of proteins with high pressures has an impact on meat qualities such as color and gel [46]. Supavititpatana and Apichartsrangkoon [7] found that gel had better quality characteristics following high pressure treatment (300700 MPa) as opposed to heat treatment. With the treatment of high pressures of 200 MPa, the chicken myobrils level increased about 120% when compared with those with no treatment [8]. Marcos et al. [9] demonstrated that high pressures (200600 MPa) induced a reduction in protein solubility compared with non-treated controls. Previous studies on WHC of meat products show that the value of WHC would increase with treatment at high pressures. Hong et al. [10] reported that the WHC of pork was signicantly decreased depending on the pressure increase (<200 MPa), while cooking loss was gradually increased. Anita et al. [11] demonstrated that pressure treatment (at 200 MPa) gave signicant reductions in cooking losses of beef batters with 1% added salt when compared with no pressure treatment. Marcos et al. [9] indicated that HPP at a pressure level higher than 200 MPa induced a reduction in WHC. Cooking loss is an important indicator of the WHC. According to previous studies [911], there is a negative correlation between cooking loss and WHC. However, the result of applying high pressures in terms of the cooking loss of raw beef remains to be elucidated. In the present study, we investigate the effects of high pressures on cooking loss, protein solubility and the histological structure of beef. The mechanism of pressure resulting in changes in cooking loss is also discussed.

2. 2.1.

Materials and methods Preparation of meat samples

The beef used for study, obtained from the local slaughterhouse (Tanggu, Tianjin, PR China), was rump, without any pretreatment (pH 5.43 0.11), and was stored at 4 C for 10 h until use. Whole muscle samples were cut into about 1.5 3.0 6.0 cm3 according to the bers parallel to the longest axis, and were sealed in barrier bags individually. All processing was performed in a food-processing laboratory at an air temperature of 10 C. 2.2. High-pressure processing and cooking Pressure treatments were performed by using a 0.6 L high-pressure vessel which was lled with water. Beef samples were subjected to various pressures from 100 to 600 MPa at room temperature for 10 min and stored at 4 C until required for analysis. Following the HPP, beef samples were cooked by heating in a water bath at 75 C for 30 min until they reached an internal temperature of 72 C and then cooled down. 2.3. Cooking loss and drip loss

The weight of beef samples before and after cooking were recorded and cooking loss was calculated for three replicates per treatment:

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Cooking loss = (m1 m2 )/m1 100%, where m1 is the the weight of the beef sample before cooking and m2 the weight of the sample after cooking. For conducting drip loss experiments, the beef samples after pressure treatment (0.1600 MPa) were weighed and placed in plastic bags for 24 h at 4 C, and then were weighed again. Drip loss parameter was determined using the relationship: Drip loss = (P1 P2 )/P1 100%, where P1 is the initial sample weight and P2 the nal sample weight.

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2.4. Measurement of pH One gram of beef sample was homogenized for 20 s at 12,000 rpm with 10 mL distilled water for 1 min and maintained for 30 min at room temperature. The pH was measured by a pH meter with a combined glass electrode.

2.5. Protein solubility The solubility of sarcoplasmic protein, myobrillar protein and total soluble protein were measured according to the method as described by Joo [12]. Sarcoplasmic protein: to 1 g of the beef sample was added 10 mL of 0.025 M ice-cold potassium phosphate buffer solution (pH 7.2) and this was homogenized for 20 s at 5000 g three times. The solution was shaken for 12 h at 4 C, then centrifuged at 1500 g for 10 min. The protein concentration levels (mg/g) were determined by biuret method [13]. Total soluble protein: to 1 g of the beef sample was added 20 mL of 0.1 M ice-cold potassium phosphate buffer solution (pH 7.2) of 1.1 M KI and this was homogenized 20 s at 5000 g three times. The solution was shaken for 12 h at 4 C, then centrifuged at 1500 g for 10 min. The protein concentration levels (mg/g) were determined by biuret method. Myobrillar protein: the solubility of myobrillar protein is calculated as the solubility of total soluble protein minus the solubility of sarcoplasmic protein.

2.6. SDSPAGE analysis The beef was triturated in liquid nitrogen and ice-cold RIPA buffer was added to extract whole-cell protein, which contained 50 mM TrisHCl (pH 7.4), 150 mM NaCl, 1% nonyl phenoxypolyethoxylethanol-40, 0.5% sodium deoxycholate, 0.1% SDS and 0.1 mM PMSF, maintained for 30 min at 4 C and centrifuged for 20 min at 10, 000 g at 4 C. A Bradford assay [14] was performed to determine the protein concentration levels. The samples were then dissolved in the SDS sample buffer solution containing 1% SDS, 1% -mercaptoethanol, 1 mM EDTA and 20% glycerin and heated for 5 min at 100 C. Twelve percent PAGE was prepared and the samples were subjected to gel electrophoresis at a constant current of 20 mA per gel. The running buffer solution contained 0.1% SDS and 25 mM Trisglycine buffer, pH 6.8. After electrophoresis, the gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250. Gel images were captured on a Gel Doc XR Gel Documentation System (Bio-Rad, Hercules, CA, USA).

2.7. Fiber fragments One gram of the beef sample was homogenized at 12,000 rpm in 30 mL of 0.25 M ice-cold sucrose solution for 30 s. One droplet of suspension was observed on a glass slide under a microscope.

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2.8. Histological staining to analyse sarcomere length by HE The beef was cut into nubs and placed in a Petri dish with 10% formaldehyde and 2% CaCl2 in phosphate buffer for more than 3 h, dipped in running water overnight, gradually dehydrated in ethanol of different strengths, dimethylbenzene, and nally embedded in parafn and processed for histological examination by microscopy (Nikon Eclipse 90i, Tokyo, Japan) after staining with hematoxylineosin [15]. The length of 200 sarcomeres was measured by Nikon Basic Research Imaging Software (Nikon, Tokyo, Japan), then calculated.

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2.9. Statistical analysis All the data in this work are representative of four experiments. Results are expressed as the meanstandard deviation of the indicated number of replicates, and differences between groups were assessed using SPSS software (Statistical Product and Service Solutions, USA).

(a) 55% Cooking loss 50% 45% 40% 35%

0.1

100

200

300

400

500

600

Pressure (MPa) (b) 5.90 pH

5.70

5.50 0.1 100 200 300 400 500 600 Pressure (MPa) (c) 11% 10% Drip loss 9% 8% 7% 6% 0.1 100 200 300 400 Pressure (MPa) 500 600

Figure 1. Changes in cooking loss, pH value and drip loss of beef with different high pressure treatments for 10 min. (a) Cooking loss of beef with high pressure treatment. (b) The pH value of beef with high pressure treatment. (c) Drip loss of beef with high pressure treatment.

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3.

Results

3.1. Cooking loss, pH changes and drip loss of beef with the treatment of high pressures As shown in Figure 1(a), there were signicant reductions in the cooking loss of beef samples treated with pressures of 300400 MPa when compared with the treatment of pressures less than 300 MPa. However, at a pressure above 400 MPa, the cooking loss of beef was close to the level of 0.1 MPa at a pressure of 500 MPa, or even higher than the level of 0.1 MPa at a pressure of 600 MPa. Our data shows that cooking loss of beef samples pressurized at 300400 MPa had a minimum of about 40.0%, which reduced by nearly 12% when compared with 0.1 MPa. As we increase the pressure from 0.1 to 500 MPa, the pH level of the beef was signicantly increased from 5.60 0.01 to 5.91 0.02. As the pressure was increased to 600 MPa, the pH level of the beef dropped to 5.85 0.02 (Figure 1(b)). Meanwhile, our data showed that the drip loss of the beef after pressure treatment at 300 or 400 MPa had a minimum of about 7.60%, as shown in Figure 1(c), which reduced by nearly 20% when compared with 0.1 MPa. 3.2. Protein solubility and SDSPAGE analysis We also found signicant changes of muscle protein solubility with high pressure treatment, as shown in Figure 2. With increasing pressure, the protein concentration of sarcoplasmic protein continuously decreased. However, both the total soluble protein content and myobrillar protein content reached the peak value at a pressure of 100 MPa. Then the concentration of total soluble protein and myobrillar protein decreased at pressures above 100 MPa. As shown in Figure 3, with the high pressure treatment of 300 MPa, the bands of myosin heavy chain and light chain began to lighten. The bands of actin during the HPP were almost unchanged. These phenomena indicate that the treatment of high pressures of over 300 MPa mainly affect the myosin protein (marked M, L in Figure 3), make it undissolvable, and result in a reduction of myobril protein (marked A in Figure 3) solubility when the pressure is over 300 MPa. 3.3. Histological structure analysis of beef The beef ber fragments were observed under microscope, as described in Section 2. We found that the ber fragments were changed signicantly from thick and short to thin and long as
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Protein concentration (mg/g)

Sarcoplasmic protein Myofibrillar protein

120

Total soluble protein

60

0 0.1 100 200 300 Pressure (MPa) 400 500 600

Figure 2. Changes in protein solubility of pressure-treated beef for 10 min. The protein concentration levels (mg/g) were determined by biuret method.

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Figure 3. SDSPAGE of total protein of beef following different pressure treatments. Lanes: beef treated with high pressures of 0.1 MPa (a), 100 MPa (b), 300 MPa (c) and 500 MPa (d). Myosin heavy chains (M), light chains (L) and actin (A) are indicated by arrows.

the pressure increased (Figure 4). This indicated that high pressure treatment might change the structure of beef muscle bers. We also calculated the sarcomere length after high pressure treatment. The sarcomere length increased as the pressure increased from 0.1 to 300 MPa (Figures 5 and 6), as expected. This indicated that the inner space of the sarcomere became larger. There was a slight decrease in the sarcomere length treated with high pressures of up to 500 MPa when compared with 300 MPa.

4.

Discussion

The aim of this study was to determine the effect of high pressures on cooking loss in beef. In addition, we shall discuss the mechanism of pressure effects on the cooking loss of beef such as changes in pH, protein solubility and histological structure. In the present work, we nd that cooking loss in beef treated at 300 or 400 MPa reduces by 12% compared with 0.1 MPa (Figure 1(a)). Previous studies by other researchers indicate that pressure treatment of raw meat products brings a small increase in pH [2], which probably results from a decrease in the acidic groups of proteins caused by unfolding. It has been known that the increase in pH after treatment will raise the WHC and reduce the cooking loss of the meat [16]. In our study, the initial pH of the raw beef was about 5.60, which then increased to about 5.91 with pressure treatment (Figure 1(b)). Pressure treatment may cause intracellular buffer solution exuding into extracellular space or change the protein structure and release alkaline groups. These would all cause a pH increase. The increase in pH with high pressure treatment will make the protein of the beef muscle deviate from the

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Figure 4. Changes in ber fragments with different pressures. (a) Pressurized at 0.1 MPa; (b) pressurized at 100 MPa; (c) pressurized at 300 MPa; (d) pressurized at 500 MPa. Magnication: 4.

intrinsic pI, enhance the repulsion between proteins and retain more water. In this work, though the changes in pH and cooking loss are inconsistent, the increase in pH with HPP does affect the cooking loss to a certain extent. Protein solubility reects the extent of protein denaturation, which also plays an important role in the WHC. In previous studies, it was indicated that the solubility of sarcoplasmic protein and myobrillar protein could affect the WHC of meat, and the changes of WHC were a result of the denaturation of sarcoplasmic protein and myobrillar protein [12]. In our study, the protein solubility changed signicantly with high pressure treatment, especially the solubility of myobrillar protein (Figure 2). A low pressure (0.1100 MPa) can promote the solubility of muscle protein, especially myobrillar protein. At pressures above 300 MPa, the solubility of myobrillar protein declined. We suggest that high pressure treatment of over 300 MPa would change the structure of the myobrillar protein, which makes it brotic and denaturalized. While the cooking loss reduced by 12% at 300 MPa (Figure 1(a)), the solubility of the myobrillar protein was reduced by more than 51% (Figure 2). This indicates that high pressures can dramatically change the structure of myobrillar protein, which cause the space between the proteins to enlarge and is helpful for maintaining the WHC of beef protein when the beef samples are cooked. It has been reported that the drip loss increased and WHC decreased because the space of bers shrunk when the sarcomere became short [1719]. Our data show that cooking loss and drip loss of beef have a positive correlation, and also that they have a negative correlation with WHC. Further, the result shows (Figures 4 and 6) that the thinner beef bers and longer sarcomeres with high pressure treatment enlarge the space between the beef bers, which is benecial for maintaining water. This suggests that cooking loss decreased gradually with high pressures increasing to 400 MPa. At an appropriate pressure (<400 MPa), slippage may occur between actin

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Figure 5. Histological staining of beef tissue by hematoxylineosin. (a) Pressurized at 0.1 MPa; (b) pressurized at 100 MPa; (c) pressurized at 300 MPa; (d) pressurized at 500 MPa. Magnication: 40.

2.5

**
Length (mm)

**
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Figure 6. Changes of sarcomere length with the treatment of high pressures of 0.1, 100, 300 and 500 MPa. p < 0.01 versus treatment of high pressure at 0.1 MPa.

myolament and myosin myolament as a result of the changes in protein structure. This process leads to the enlargement of the sarcomeres inner space, which can increase the WHC. Moreover, macromolecules may degrade into micromolecules. This means the surface of the molecule increases, and thus has more capacity to hold water. However, excessive pressures of 500 and 600 MPa might provoke beef bers to become overstretched and fractured, ruining the structure of the sarcomeres. These consequences might cause leakage of tissue uid, eventually leading to the level of WHC declining.

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High pressure treatment can maintain a products nutritional properties. It also increases food safety and reduces cooking loss without the use of chemicals or irradiation, and further creates no byproducts. Moreover, high pressure treatment may also extend the shelf-life of products. As high pressure treatment has many prominent advantages, we believe that it will be extensively applied in the meat product industry in the future. References

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