Technical Paper
Comparison of Plant-origin Proteases and Ginger Extract on Quality Properties of Beef Rump Steaks
2019 年 25 巻 4 号 p. 529-538
詳細
2019 年 25 巻 4 号 p. 529-538
The objective of this study was to compare the effect of papain, bromelain, kiwifruit protease, ficin and ginger extract with different concentration (0, 20, 40, 60, 80, 100 U/g) for improving the quality properties of beef rump steaks. The pH, cooking loss, Warner–Bratzler shear force, texture, colour, myofibrillar fragmentation index (MFI), collagen heat solubility, content of soluble protein (CSP), electrophoresis and sensory evaluation are invested. The result showed that all of plant-origin proteases and ginger extract can improve quality characteristics of rump steaks, and ginger extract was found to be the most effective at reducing cooking loss and contributing to the desirable red color and sensory properties, while papain and bromelain were found to be the most effective at decreasing Warner–Bratzler shear force. Ginger extract can be applied in the industrial scale and household level as a convenient and effective method to improve meat qualities. Furthermore, high-quality rump beef steaks at a keen price would be more available for Chinese consumers.
In recent years, with the improvement of Chinese living standard and the need for food diversity, beef steaks are enjoying an increasing popularity by its characteristics of nutritious, convenient and delicious. However, steaks are always too expensive to satisfy the requirement of common consumers, and the meats for steaks are always in high qualities. So it's necessary to make a deep research on the quality improvement of ordinary steaks. Tenderness, flavor, and juiciness are the top three characteristics that affect the sensory quality of meat and meat products (Aaslyng and Meinert, 2017), besides, color and texture properties are also important indicators. While the most important factor to determine consumer's repeat purchase is tenderness (Mennecke et al., 2007). Nowadays, the research about meat quality improvement mainly focused on tenderness enhancement and various enzymatic, physical and chemical approaches have been applied (Zhou et al., 2015; Hoffman et al., 2008; Abdel and Mohamed, 2016), and the effectiveness of enzymatic approach has been testified by many researchers. When actinidin from Chinese kiwifruit cultivars was used for Pork and rabbit longissimus dorsi, the reduced shear force by more than half was recorded (Zhang et al., 2017). A significant decrease in filtering residue, Warner-Bratzler shear force and textural parameters was recorded when papain (0.1% enzyme solution) was applied to beef longissimus lumborum (Barekat and Soltanizadeh, 2016). There was a significant increase in collagen solubility and sensory scores when ginger, papain and their mixture were added to Camel meat burger patties, and a significant reduction in the shear force was also recorded (Abdel-Naeem and Mohamed, 2016). Manohar et al. found that bromelain (1%, 2%, 3% and 4%) can improve the tenderness of red meat (2016). Ha et al. (2012) hydrolyzed beef connective tissue and myofibrillar extract using papain, bromelain, actinidin and zingibain protease, and the kiwi protease was found to be most effective at hydrolysing myofibrillar extract, while ginger protease the most effective at collagen. Previous studies have demonstrated that a variety of plant-origin proteases have a good tenderization effect on meat and meat products (Botinestean et al., 2017; Aminlari et al., 2010; Naveena et al., 2004; Ramezani et al., 2003), and it is also reported that ginger extract is more efficient at hydrolyzing collagen, which is an important factor contribute to the hard texture of meat (Kim et al., 2007). However, there are few reports when it comes to characteristics like tenderness, texture properties, color, sensory scores and protein hydrolysate of samples when treated by different proteases. We are wondering if there is a tenderizer that can improve the overall properties including tenderness, color, flavour etcetera. The present study analyzed the effect of papain, bromelain, kiwifruit protease, ficin and ginger extract with different concentration on bovine buttocks. The changes of pH, cooking loss, shear force, color, texture properties, myofibril fragmentation index, collagen solubility and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were recorded, which provide a comparative and promising approach to the improvement of the quality of bovine buttocks steaks.
Materials All of the beef samples collected for the experiment were rump beef muscles (Changchun Haoyue Co., Ltd., China), which were selected from 18- to 24-month-old Simmental cross bred oxen (weight 400–500 kg). After slaughter, the carcasses were chilled for 24 h at 4 °C, then divided and transported to the laboratory. Papain, bromelain, kiwifruit protease, and ficin were purchased from Henan Shengside Ltd., China and Fresh gingers from local supermarket. Fresh ginger were peeled, sliced, and blended in the liquidizer for 2 min, the homogenate were filtered with four layers of muslin cloth, and the filter were collected as ginger extract. All the chemicals and reagents used were of analytical grade.
Sample preparation The fat and connective tissues on the muscles were removed carefully, and the meat was cut into steaks of 2 cm×5 cm×10 cm in size. Papain, bromelain, kiwifruit protease, ficin and ginger extract were diluted into 20 U/g, 40 U/g, 60 U/g, 80 U/g, 100 U/g and the enzyme and ginger extract solutions were adjusted to their optimum pH with citric acid solutions, papain 6.0, bromelain 6.0, ficin 7.0, kiwifruit protease 7.5, ginger extract 6.0 (Arshad et al., 2016; Yamaguchi et al., 1982; Thompson et al., 1973).
Steaks prepared were divided into 5 groups, each group was injected with 10% papain, bromelain, kiwifruit protease, ficin and ginger extract solutions of different enzyme activity uniformly with a syringe manually. The samples were placed into polyethylene plastic bags and stored under 30 °C for 2 h, then the analysis of all treatments were conducted.
Measurement of proteolytic activity The proteolytic activity of enzymes and ginger extract was determined before dilution, and the determination procedure performed according to Bruno et al. (2010) with some modification. Briefly speaking, 1 mL protease solution was mixed with 1 mL casein solution (1 g/100 mL) in 0.1 mol/L sodium phosphate buffer (pH 7.5). The mixture was incubated for 10 min in Water-Bath at 4 °C, and the reaction stopped by the addition of 2 mL of trichloroacetic acid (TCA, 65.4 g/L). Blanks were prepared by adding TCA to the enzyme, then the casein solution. The solution was filtered with filter paper, and filtrate was collected and mixed with folin phenol for the detection of absorbance at 280 nm.
Measurement of Cooking loss The samples were cut into pieces of 2.5 cm3, and each was weighted accurately as w1. Cooking the sample at 80 °C in water-bath until the inner temperature reached 70 °C. The sample were cooled by running water and wiped with blotting paper and weighted accurately again as w2. The cooking loss was expressed as the percentage of w1 and the difference between and after cooking.
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Measurement of Warner–Bratzler shear force The cooked samples was chilled at refrigerator for 12 h, six meat columns with 1.27 cm diameter were taken from each sample paralleling to the muscle fiber orientation by sampler. Warner–Bratzler shear force of each column was determined using the tenderness meter (RH-N50, Guangzhou Runhu Instrument Co., Ltd., China). Warner–Bratzler shear force was expressed by the average peak force of six columns.
Measurement of Texture properties The cooked sample was chilled and cut into pieces of 1 cm3. The determination of texture properties was performed though CT3-50 kg texture analyzer (Brookfield Engineering Labs, Inc., 11 Commerce Boulevard, Middleboro, 02346 U.S.A.). Hardness, elasticity and chewiness were evaluated by TA39 probe. Every determination was repeated at least three times and the average value were recorded.
Color evaluation The color evaluation was performed through the CIE L* a* b* system of WSF spectrophotometer (INESA Instrument Co., Ltd., Shanghai). The colorimeter was standardized by calibration board before evaluation, and parameter lightness (L*), redness (a*), yellowness (b*) were recorded, three replicates from the surface of the sample were carried out for each samples and the average value were recorded.
Measurement of MFI MFI was determined according to Culler et al. (1978) with little modification. Two grams of minced meat (raw) was blended with 20 mL ice-cold buffer (100 mM potassium chloride, 7 mM monopotassium phosphate, 18 mM dipotassium hydrogen phosphate, 1 mM disodium ethylenediamine tetraacetic acid and 1 mM magnesium chloride, pH 7.0.) at centrifuge tube, homogenized at 8 000 rpm for 30 s. The homogenate was centrifuged at 5 000 rpm for 15 min at 4 °C. The suspension was abandoned, and 20 mL ice-cold buffer was added to the tube again, and the homogenization and centrifugation procedures were repeated. The precipitation was kept and 5 mL ice-cold buffer was added and homogenized again, then connective tissues and debris were removed by a 20-mesh screen filter, and the filtrate was kept as the myofibril extract.
The protein concentration of the myofibril extract was determined by the biuret method according to Gornall et al. (1949). The protein concentration was diluted to 0.5 ± 0.05 mg/mL with a separation medium. The diluted samples were poured into the cuvette immediately after stirring and the absorbance is measured at 540 nm by UV spectrophotometer (UV-8000, Shanghai Yuanxi Instrument Co., Ltd. China). Every sample was repeated three times, and MFI was expressed as the mean absorbance multiplied by 200.
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Measurement of Collagen Heat Solubility Collagen heat solubility was determined according to Naveena et al. (2011) with few modifications. Total collagen content preparation: 1 gram of sample and 25 mL sulphuric acid solution (3 mol/L) was added into a conical flask and sealed by aluminium foil, hydrolyzed at 108 °C for 18 h. The hydrolysate solution was filtered into a 25 mL volumetric flask while it is hot, and the beaker and filter paper were washed several times by sulphuric acid solution, and transformed into volumetric flasks, the volume was adjusted to 25 mL with distilled water. Insoluble collagen content preparation: 1 gram of sample was blended with 15 mL 1/4 Ringer's buffer (1.8 g sodium chloride, 0.25 g potassium chloride, 0.06 g calcium chloride hexahydrate, 0.05 g calcium chloride hexahydrate, 0.186 g acetic acid, diluted in 1 L distilled water) in centrifuge tube, homogenized and kept in water bath at 77 °C for 60 min, centrifuged at 4500 r/min for 15min when it's cold. The precipitate was hydrolyzed with 20 mL sulphuric acid solution (3 mol/L) at 108 °C for 18 h. The hydrolysate solution was filtered, transformed, and adjusted in a 25 mL volumetric flask as procedures in total collagen.
The content of hydroxyproline in meat and meat products was determined the method described by Mahendrakar et al. (1989). Collagen content is equal to hydroxyproline content multiplied 7.14 (Naveena et al., 2011).
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CSP and Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 2 grams of minced samples was mixed and homogenized with 20 mL ice-cold extract buffer (100 mM potassium chloride, 20 mM tripotassium phosphate, 1 mM calcium chloride, 1 mM ethylene diamine tetraacetic acid). The mixture was centrifuged and the supernatant was collected for the determination of CSP and SDS-PAGE analysis. The CSP was determined by biuret method and diluted to 3.0 ± 0.5 mg/mL with 1.0 M Tris-HCl (pH 7.0) buffer. 20 µL diluted protein solution was mixed with 5 µL buffer (distilled water 2 mL, 1 M Tris-HCl (pH6.8) 300 µL, bromophenol blue 0.005 g, SDS 0.100 g, glycerol 2.5 mL, 250 µL M DTT), heated at boiling water for 5 min. 5 µL sample was used for loading in the gel which is formed by a 5% stacking gel and the 10.5% separating gels. Electrophoresis was performed at constant voltage 80 V/slab for 3 h and transformed to 100 V/slab for 4–5 h. Finally, the gels were removed, stained, decolored and photographed. The molecular weight of each protein bands of our samples were estimated according to the molecular weight markers, and bands of myosin (200 kDa), β-galactosidase (116 kDa), phosphatase b (97.2 kDa), bovine serum albumin (66.4 kDa), ovalbumin (44.3 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20.1 kDa), lysozyme (14.3 kDa) and aprotinin (6.5 kDa) were contained in the marker.
Sensory evaluation Cooked steaks treated by enzyme and ginger extract (40 U/g) were evaluated by seven trained assessors (graduate students majored in food science in Jilin University). An 8–point descriptive scale (where in 1 is extremely undesirable and 8 is extremely desirable) was used to assess the tenderness, juiciness, flavor, appearance and overall acceptability (Keeton, 1983). Water was provided to panelists to cleanse their palate between samples.
Statistical analysis All experiments were conducted in triplicate and the results were expressed as means ± standard deviation (SD). Origin 9.1 software was used to process graphs. Duncan's multiple range tests were used to analyze the variance (ANOVA) to determine the results' significance at p < 0.05, using SPSS 19.0 software (SPSS Inc. Chicago, USA).
The change of cooking loss As an important parameter of meat quality, cooking loss indicates the fat retention ability and moisture retention ability during the cooking process (Abdel-Naeem and Mohamed, 2016). The cooking loss of the steak samples treated with different concentration of proteases and ginger extract are shown in Fig. 1. There is a significant increase of cooking loss in all treated samples with the increase of enzyme concentration before 60 U/g. Previous studies have recorded a similar conclusion, Istrati et al. (2012) and Botinestean et al. (2017) reported a higher cooking loss of samples when treated with papain and bromelain. The cooking loss of all treated samples are reached a peak value when the enzyme concentration is 60 U/g, and the highest value of 36.19% were reached by papain treatment samples, followed by bromelain treatment samples of 35.91%, and the lowest value of 29.33% was ginger extract treatment samples. Abdel and Mohamed (2016) also found that the camel meat burger patties treated by papain achieved a higher cooking loss, while patties treated by ginger extract achieved a lower cooking loss. This is disagree with Ruitong et al. (2010) who reported an increase of cooking yield of yak meat after treatment with ginger extract. A research conducted by He et al. (2015) showed that ginger extract has no significant effect on cooking loss of duck breast. The cooking loss of all of the treated samples are decreased significantly when the enzyme concentration was over 60 U/g, and the drop of the cooking loss may result from the over-tenderization of muscles, and the moisture and fat are retained by the excessively hydrolyzed muscles during the cooking process (Abdel-Naeem and Mohamed, 2016).
Effect of different plant-origin proteases and ginger extract on cooking loss
The data is represented as mean ± S.D. (n=3)
The change of Warner–Bratzler shear force Warner–Bratzler shear force can be the most effective criterion to evaluate meat tenderness. Results of Warner–Bratzler shear force changes after treatment with different plant - origin proteases and ginger extract are presented in Fig. 2. The Warner–Bratzler shear force of all the treated samples are decreased dramatically with the increase of enzyme activities. Steak tenderness may be enhanced by the hydrolysis of connective tissue proteins and myofibrillar proteins under the function of enzymes, and the muscle structures were also destroyed (O'Meara and Munro, 1985). Previous studies, also reported similar results regarding effects of protease on meat tenderness. Christensen et al. (2009) has reported a significant decrease of shear force of porcine M. biceps femoris when actinidin was injected. A significant reduction of shear force values of yak meat, buffalo meat and duck breast muscles were also recorded when ginger extract was applied (Ruitong et al., 2010; Naveena and Mendiratta, 2010; He et al., 2015).
Effect of different plant-origin protease and ginger extract on Warner–Bratzler shear force
The data is represented as mean ± S.D. (n=3)
It can also be concluded that the higher enzyme concentration, the lower Share force values. After the treatment of plant-origin protease and ginger extract with 100 U/g, the share force values for ficin group, kiwifruit protease group, papain group, bromelain group, and ginger extract group were decreased by 74%, 76.6%, 78.5%, 76% and 71% respectively, which indicates that ficin, kiwifruit protease, papain, bromelain, and ginger extract are effective at tenderizing bovine buttocks muscles. However, the over-tenderization effect happened when steaks treated with ficin, kiwifruit protease, papain, bromelain over 80 U/g, and both the appearance and morphological integrity of the rump steaks were destroyed. At the same time, the shear force was unsuitable to be the evaluation index of the samples quality anymore.
Texture properties The effect of different plant-origin proteases and ginger extract on texture properties of steaks can be seen in Fig. 3. Hardness refers to the internal binding force that keeps the shape of an object. The higher the hardness, the lower the tenderness, and hardness also related to sensory chewiness. Hardness values decreased significantly in rump steaks treated by plant-origin proteases and ginger extract. When the enzyme activity was 60 U/g, the hardness of ficin group, kiwifruit protease group, papain group, bromelain group, and ginger extract group decreased by 67%, 71%, 61%, 72% and 51%, respectively. With further increase of enzyme activity, the hardness continues to decrease, but the morphological integrity of the steak samples was destroyed under the hydrolysis of the proteases, and the undesirable characteristics appeared. Similar decrease in hardness of squid (Gokoglu et al., 2016), porcine biceps femoris muscles (Christensen et al., 2009), beef steaks (Botinestean et al., 2017), beef and chicken (Eom et al., 2015) were also recorded.
Effect of different plant-origin proteases and ginger extract on texture properties: a-Hardness, b-Springiness, c-Chewiness
The data is represented as mean ± S.D. (n=3)
Springiness is the ability to recover to the original shape when the external force was removed. As shown in Fig. 3b, the springiness of all treatment groups decreased with the increase of enzyme activity. While the springiness of the ginger extract treatment group increased firstly and then decreased. When the enzyme concentration was 40 U/g, the springiness reached a peak of 3.44 mm. Ginger extract can have the ability to increase the water holding capacity of rump steaks, thus contribute to the increased springiness. With further increase of enzyme activities, the myofibrils are hydrolyzed, and the water within the internal structures was lost, which result in the decrease of springiness. The chewiness of all treated samples decreased significantly with the increase of enzyme activities (Fig. 3c). The samples are over-tenderized when treated with enzymes of high activities and the original internal morphological structure was destroyed. Accordingly, the hardness, springiness, and chewiness are decreased rapidly. When the enzyme concentration was 100 U/g, the springiness of all of the five groups dropped to its lowest level, and the ginger extract treatment group has the highest springiness of 2.31 mm and the papain group the lowest of 1.41 mm among the five groups (P < 0.05).
Color The changes of the L*, a* and b* values can be seen in Table 1. Insignificant change of L* values can be seen in ficin treated groups, kiwifruit protease groups and bromelain groups, and the b* values of treated samples showed no remarkable difference in ficin and kiwifruit protease treated samples but significantly higher than control, and there's no significant change of b* value when bromelain and papain applied. The a* value of groups treated by four proteases decreased significantly as increase of enzyme activity (P < 0.05). While previous research showed that there is no significantly (P < 0.05) difference in L* a* and b* values of papain treated samples when compared with control samples (Abdel-Naeem and Mohamed, 2016). Nadzirah et al. (2016) found that the L* and b* values of bromelain–treated beef were significantly higher than control and the a* value was lower. Botinestean et al. (2017) also recorded a lower L* and b* values of beef steaks when treated with papain, bromelain and a mixture of them (1:1), and there's no significant change when it comes to a* values. A significant increase of a* value can be seen in ginger treated samples, which means the more ginger extract, the higher red colors of beef muscles. This could be due to the antioxidant activity of ginger extract which protecting color by delaying the oxidation of myoglobin and retarding the formulation of metmyoglobin. Mansour et al. (2000) found out that ginger rhizome extract exhibited strong antioxidant activity and patties prepared with it had significantly higher red colors, which is consistent with our research. However, research indicates a non-significant (P < 0.05) difference in L*, a* and b* values in ginger treated chicken emulsion in the end of storage (Singh et al., 2014). Mancini et al. (2017) recorded a reduced a* value and an increased b* value of pork burgers when treated with ginger power, and the difference can result from the curcumin in ginger power.
| Enzyme Activity | Ficin | Kiwifruit protease | Bromelain | Papain | Ginger extract | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| U/g | L* | a* | b* | L* | a* | b* | L* | a* | b* | L* | a* | b* | L* | a* | b* |
| 0 | 22.38±1.67 | 32.65±1.38a | 25.77±0.55b | 22.38±1.67 | 32.65±1.38a | 25.77±0.55b | 22.38±1.67 | 32.65±1.38a | 25.77±0.55 | 22.38±1.67b | 33.32±1.94a | 25.77±0.55 | 22.38±1.67ab | 32.65±1.38b | 25.77±0.55a |
| 20 | 21.84±1.21 | 32.20±0.80ab | 27.59±0.67a | 22.49±2.05 | 30.93±0.03ab | 27.59±0.67a | 22.78±2.04 | 32.24±2.17a | 27.09±1.33 | 24.65±0.99ab | 31.47±2.16ab | 25.98±1.04 | 24.30±0.63a | 32.76±1.29b | 24.00±0.70b |
| 40 | 21.18±1.36 | 31.42±1.26ab | 28.40±1.42a | 23.37±1.23 | 30.57±0.94b | 28.40±1.42a | 23.39±1.50 | 29.94±2.08ab | 27.21±2.18 | 23.89±1.65b | 28.53±1.36b | 26.41±0.93 | 22.36±0.66ab | 33.00±0.77b | 23.6±1.14b |
| 60 | 21.59±0.76 | 30.97±1.99ab | 28.06±1.00a | 22.91±1.94 | 28.92±1.52bc | 28.06±1.00a | 24.33±1.54 | 28.87±1.04b | 27.41±1.33 | 25.02±0.70ab | 28.56±2.19b | 26.92±0.35 | 20.54±0.74b | 33.24±0.70b | 23.09±1.28b |
| 80 | 20.79±1.16 | 29.83±2.18b | 28.52±0.86a | 23.65±1.78 | 28.48±1.49c | 28.52±0.86a | 23.91±1.94 | 28.28±1.02b | 27.07±1.53 | 25.66±1.42ab | 27.46±1.34b | 26.32±1.12 | 20.02±1.55b | 35.21±0.36a | 24.09±0.73ab |
| 100 | 21.13±1.09 | 28.95±0.52b | 27.55±0.75a | 23.16±1.92 | 27.63±0.64c | 27.55±0.75a | 24.56±1.00 | 28.13±1.61b | 27.37±0.51 | 27.15±1.82a | 27.41±0.85b | 25.21±1.70 | 20.00±1.13b | 36.14±0.67a | 24.00±1.26b |
Superscripts with different letters in the same column indicate significant differences (p < 0.05).
Mean ± S.D. (n = 3).
Myofibril fragmentation index Myofibril fragmentation index is an important indicator of beef tenderness and associated with the degradation of myofibril (Olson, 1977). The MFI change of steaks treated by different protease and ginger extract of different concentration can be seen in Fig. 4. Generally, the MFI values of all treated samples were increased significantly with the rise of enzyme activities. The hydrolysis of actin and myosin driven by protease tenderization can be a contributable factor, and Z-lines were breakdown, which may lead to the shortening of myofibril length and the reduction of sarcomere number. All of these reactions contributed to a damage of myofibrils and the fragmentation progress were accelerated. The result was consistent with previous studies. A significant increase of MFI values of duck breast muscles and bovine longissimus dorsi muscle were recorded when ginger extract and Sarcodon aspratus extract were added respectively (He et al., 2015; Shin et al., 2008). Gerelt et al. (2000) also reported a significant increase of MFI value of cow meat when treated with papain and proteases from Aspergillus sojae and A. oryzae. The MFI of ficin treated samples and bromelain treated samples reached a peak of 127.2 and 119.5 respectively when the enzyme activity was 80 U/g. With further increase of enzyme activity, the MFI showed a downward trend, but the difference is not significant (P > 0.05). The difference might result from the higher myofibril hydrolyzing ability of ficin and bromelain, and a dramatically increase of myofibrillar proteolysis was appeared when treated with enzyme of 80 U/g (Ha et al., 2012). Finally, the fragmentation degree of myofibrils treated with different proteases was: ficin treatment group (138.2) > papain treatment group (125) > bromelain treatment group (124.5) > ginger extract treatment group (116.6) > kiwifruit protease treatment group (110). Different proteases have different proteolytic activities toward myofibrils can be a reason for the difference.
Effect of different plant-origin proteases and ginger extract on MFI
The data is represented as mean ± S.D. (n=3)
Collagen Heat Solubility As shown in Fig. 5, enzymes were able to increase the collagen heat solubility of rump steaks significantly. The collagen heat solubility ranged from 16% to 68% when enzymes were injected and the higher the enzyme activity, the greater the heat solubility of collagen. The collagen heat solubility increased dramatically when the enzyme activity raise from 20 U/g to 80 U/g and the hydrolysis rate of collagen slowed down with further increase of enzyme activity. The collagen heat solubility of samples treated by ficin, kiwifruit protease, bromelain, papain and ginger extract were 60%, 53%, 63%, 65% and 68%, respectively, when the enzyme activity was 100 U/g, which indicates that ginger extract appears to be the most effective at hydrolysing collagen and connective tissue proteins. The result was consistent with Ha's research about the commercial papain, bromelain, actinidin and zingibain and their activities toward meat proteins (Ha et al., 2012). Previous research also found that there are two cysteine proteases from ginger, GP2 and GP3. With the presence of proline-containing, peptides GP2 shows preference for proline in the P2 position, and at least 10-fold higher efficiency of hydrolysis than papain, which means it can hydrolyze type I collagen effectively and increase collagen heat solubility (Kim et al., 2007).
The collagen heat solubility of samples treated by different plant-origin proteases and ginger extract
The data is represented as mean ± S.D. (n=3)
CSP and SDS–PAGE The CSP of samples treated with four proteases and ginger extract in various concentration can be seen in Fig. 6. As we can see from the figure, all of papain, bromelain, kiwifruit protease, ficin and ginger extract have a significant impact on the CSP of samples. Starting from 54.8, the CSP of samples increased significantly (P < 0.05) when treated with proteases and ginger extract, and the higher enzyme activity, the higher CSP values. When samples treated with proteases and ginger extract of 100 U/g, the CSP of all samples peaked at their highest value, and finally, there's no significant difference between samples treated by papain and ficin (P > 0.05), and CSP of samples treated by papain, ficin and bromelain were significantly higher than kiwifruit protease and ginger extract treated samples (P < 0.05).
The content of soluble protein (CSP) of samples treated by different plant-origin proteases and ginger extract
The data is represented as mean ± S.D. (n=3)
A representative protein pattern by SDS–PAGE for the steak samples treated with different concentrations of four proteases and ginger extract can be seen in Fig. 7. Proteases and ginger extract of different concentration had significant impacts on the protein patterns of rump steak samples. Similar protein patterns in samples treated by ficin and kiwifruit were observed in Fig. 7 (a) and Fig. 7 (b), high molecular weight protein bands (97.2–116 kDa) were hydrolyzed gradually as the increase of enzyme activity, which resulted in increased concentration of lower molecular weight protein bands (6.5–20.1 kDa). In Fig. 7 (c) and Fig. 7 (d), similar hydrolysis of higher molecular weight protein bands (97.2–116 kDa) can be seen when papain and bromelain applied and the higher enzyme activity, the more obvious hydrolysis effect, but there's no lower molecular weight protein bands between 6.5 and 20.1 kDa. The breakdown of proteins in the ginger extract treated samples of higher enzyme activity was clearer than others, which indicates more pronounced proteolysis. Increased proteolysis in proteases and ginger extract treated samples can be related to significantly higher protein solubility. Similar results were reported by different researchers (He et al., 2015; Kim et al., 2007; Tsai et al., 2012). Naveena found proteases from Cucumis trigonus Roxb (Kachri) was more effective than papain and ginger extract at hydrolizing proteins with high molecular weight (Naveena et al., 2004).
The SDS-PAGE of samples treated by plant-origin proteases and ginger extract
Column M: Marker; Column1: Control; Column 2: 20 U/g; Column 3: 40 U/g; Column 4: 60 U/g; Column 5: 80 U/g; Column 6: 100 U/g.
(a) Samples treated by ficin; (b) Samples treated by kiwifruit protease; (c) Samples treated by papain; (d) Samples treated by bromelain; (e) Samples treated by ginger extract.
Sensory Evaluation Table 2 showed the results of sensory evaluation in response to the different treatments. The mean scores for tenderness did differ significantly between control and treated samples, and samples treated by papain and bromelain achieved higher score and samples treated by ginger extract relatively lower, which is consistent with the result of Warner–Bratzler shear force. The highest score of flavour, appearance, and overall acceptability was achieved by steaks treated with ginger extract, which was significantly different from the other treatment samples (p < 0.05), and it may be result from the unique favorable flavor and color preservation effect of ginger extract. Although the cooking loss of treated samples was higher than control, the higher juiciness was still found in treated samples, the salt and seasoner added during the cooking process may contribute to the water holding capacity of samples. Besides, tenderness, flavour and overall acceptability of sample would have an impact on people's attitude toward the juiciness of samples.
| Sensory Qualities | Control | Ficin TS | Kiwifruit TS | Papain TS | Bromelain TS | Ginger TS |
|---|---|---|---|---|---|---|
| Tenderness | 5.27±0.10a | 6.64±0.08b | 6.71±0.07bc | 6.84±0.08c | 6.85±0.06c | 6.60±0.05b |
| Juiciness | 6.27±0.06a | 6.72±0.13bc | 6.72±0.14bc | 6.66±0.09b | 6.79±0.11bc | 7.00±0.07c |
| Flavour | 6.01±0.19a | 6.20±0.19a | 6.22±0.10a | 6.18±0.22a | 6.21±0.10a | 6.58±0.11b |
| Appearence | 6.43±0.15a | 6.37±0.15a | 6.42±0.10a | 6.39±0.12a | 6.30±0.2a | 6.70±0.18b |
| Overall acceptability | 5.58±0.16a | 6.44±0.08b | 6.49±0.13b | 6.56±0.07b | 6.58±0.16b | 6.97±0.11c |
Ficin TS= Ficin Treatment Samples, kiwifruit TS= kiwifruit Protease Treatment Samples, Papain TS= Papain Treatment Samples, Bromelain TS= Bromelain Treatment Samples, Ginger TS= Ginger extract Treatment Samples
Sensory scores based on 8 point scales:
Tenderness: 8= extremely tender, 1= extremely tough.
Juiciness: 8= extremely Juicy, 1= extremely dry.
Flavour: 8= extremely enjoyable, 1= extremely unenjoyable.
Appearance: 8= extremely tender, 1= extremely tough.
Overall acceptability: 8= extremely desirable, 1= extremely undesirable.
Mean ± S.D. (n = 7).
In addition, there was no significant difference in terms of tenderness, juicy, flavor, appearance and overall acceptability among samples treated with ficin, kiwifruit protease, papain and bromelain. In conclusion, both ginger extract and different plant-origin proteases can improve the sensory properties of beef rump steaks significantly. An improved sensory quality of chevon, beef and camel meat burger patties were recorded when ginger extract was applied (Pawar et al., 2010; Naveena et al., 2010; Abdel et al., 2016). Gokoglu et al. (2016) also reported an improved sensory quality of squid when papain and pineapple were added.
The injection of ficin, kiwifruit protease, papain, bromelain and ginger extract solutions to beef rump steaks can result in significant increase of MFI, collagen heat solubility, tenderness, and sensory quality, and the overall quality of steaks can also be improved. The effect varies when samples were applied with different enzymes, the color-protection effect was recorded when ginger extract was added, and the higher the concentration, the more desirable red color appeared, while when samples treated with other proteases, the a* value declined significantly. Ginger extract was also found to be the most effective at reducing cooking loss, hydrolyzing collagen protein and contributing to the desirable sensory properties when compared with other plant origin proteases, while papain and bromelain were found to be the most effective at decreasing Warner–Bratzler shear force. In short, ginger extract can be used to improve the quality of rump beef products as an effective substitute of ficin, kiwifruit protease, papain, bromelain. Moreover, ginger extract in proper concentration can also be applied in the industrial scale and household level as a convenient and effective method to improve meat qualities including color, tenderness, flavour and so on.
Acknowledgments The authors thank China Meat Products Research Center for the financial support during this research, and the staff of the Laboratory in Food Science and Engineering of Jilin University for their support throughout the experiments.
