Food Measure DOI 10.1007/s11694-016-9403-3
ORIGINAL PApER
Effect of bromelain and papain enzymes addition on physicochemical and textural properties of squid (Loligo vulgaris) Nalan Gokoglu1 · Pınar Yerlikaya1 · Ilknur Ucak1 · Hanife Aydan Yatmaz2
Received: 29 April 2016 / Accepted: 29 August 2016 © Springer Science+Business Media New York 2016
Abstract The aim of this study was to investigate the effects of bromelain and papain enzymes for tenderizing of squid (Loligo vulgaris) muscle. Squid samples were immersed into the enzyme solutions and then they were kept in a water bath at 60 °C for 20 min to activate enzymes. After enzyme treatment, pH values, hardness and shear force values of squid muscle decreased. Total protein, free amino acid contents and sensory scores increased. Use of bromelain and papain enzymes was found as effective method to tenderize of squid muscle. Better results were obtained with papain compared to bromelain. Keywords Squid · Texture · Tenderization · Enzyme · Bromelain · Papain
Introduction Squid is the most important member of cephalopods. It has commercial importance and high quality protein. Hardness and softness of meat affect considerably acceptability by the consumers. Cephalopod muscles are most important in this regard. Squid mantle texture is known to be related to its particular structure [1]. Essentially, muscle fibres show both radial and circular arrangement; in addition, they are
Nalan Gokoglu
[email protected] 1
Faculty of Fisheries, Akdeniz University, Antalya, Turkey
2
Food Safety and Agricultural Research—Development Center Antalya, Akdeniz University, Antalya, Turkey
supported by connective tissue with longitudinal, radial and circular orientations [1]. Proteins and their functions are the most important factors affecting texture. Proteins can be modified by various technological processes and texture can be improved [2]. Different tenderization methods such as cooking [3], addition of phosphates salts and sodium chloride [4, 5] and marination with organic acids [6] have been used. Use of proteolytic enzymes is popular for meat tenderization. Proteolytic enzymes derived from plants such as papain, bromelain and ficin have been widely used as meat tenderizers [7–10]. Papain enzymes are derived from the unripe Carica papaya L. species. The most powerful, dynamic papain enzyme could be obtained from the immature, green papaya fruit. When the fruit mature, enzymes naturally are destroyed, and the function is off [11]. Papain is regarded as natural proteolytic enzymes which could break off the lysine, phenylalanine and arginine peptide bond in the muscle protein upon tenderization mechanism [12]. Bromelain is a complex mixture of substances that can be extracted from the stem and core fruit of the Bromeliaceae or pineapple family, mostly from Ananascomosus Merr., sp. [13]. Similar to papain, bromelain enzyme soften the toughness of meat by solubilise the sarcolemmic collagen up to 40 % before attack the myofibrils of the protein [14]. In the previous studies bromelain and papain were found effective on beef [15–18], pork and chicken [16] and duck [19]. There is in limited number study related to effects on squid muscle. The aim of this study was to investigate the effects of bromelain and papain enzymes addition on physicochemical and texture properties of squid (Loligo vulgaris).
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Materials and methods
Cooking loss
Material
Weight of squid samples was measured before and after cooking to determine cooking losses, which was expressed as the cooked weight subtracted from uncooked weight, divided by the uncooked weight then multiplying by 100. Cooking loss was expressed as per cent.
Squid (Loligo vulgaris) was obtained from the main fish market of Antalya. They were purchased just after landing and placed in cold storage bag with ice. Before treatments head, skin, viscera and tentacles of squid samples were removed and then cut into square pieces of 4 × 4 cm. Commercial bromelain (from pineapple Sigma B4882) and papain (from papaya Sigma P4762) were purchased.
Enzyme application Preliminary trials were conducted to find the most proper enzyme concentration. For preliminary trials squids were dipped into papain and bromelain solutions in different concentrations. At last, concentrations of 0.001 and 0.004 % were determined as the best in terms of textural properties. While concentration lower than 0.001 % was not effective, higher ones than those of 0.004 % caused extremely soft texture, squid meat almost melted. Two solutions (0.001 and 0.004 %) of each enzyme were prepared using distilled water. After dipping into enzyme solutions squid pieces were kept in a water bath at 60 °C for 20 min to activate enzymes. Ratio of sample/solution was 1/1 (g/ml). Control samples were dipped into distilled water without enzyme. After all treatments analyses were conducted.
Analyses pH measurement Measurement of pH was carried out by dipping pH-meter probe into a mixture of homogenised squid meat and distilled water (1/1) [20]. All measurements were performed at room temperature (20 °C) using pH-meter (WTW Inolab, Weilhem, Germany). Water holding capacity Samples (1 g) were placed in polycarbonate centrifuge tubes and heated to 90 °C in a water-bath for 1 h. Samples were removed from the water-bath, cooled and centrifuged at 4000×g for 1 h (4 °C). After centrifugation, samples were dried on a blotting paper and then re-weighed [21].WHC was calculated using the following equation [21]:
[1− (first weight − last weight / moisture content of the sampless) ×100].
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Total soluble protein Total soluble protein analysis was carried out according to Lowry’s method [22] using bovine serum albumin standard. A 3 g sample was blended with 27 ml of 5 g/100 ml (%5) trichloroacetic acid (TCA) using an ultraturrax homogenizer (IKA Labortechnic, Staufen, Germany) and filtered. A 1 ml aliquot was transferred into test tubes and 1 ml lowry reagent (40 ml distilled water added into reagent and dissolved in ultrasound batch) was added. The tubes held in room temperature for 20 min. Then 0.5 ml Folinciocalteu (18 ml 2N Folin + 10 ml distilled water + 80 ml distilled water) was added and tubes were stored at room temperature for 30 min. The supernatant was then transferred to a spectrophotometric cell. The absorbance of the mixture was measured using spectrophotometer (Evolution 160 UV–visible; Thermo Scientific Dreieich, Germany) at 750 nm. Total free amino acid The sample (2 g) was mixed with 17 mL of 0.2 mol L−1perchloric acid and placed in an ultrasonic bath (GFL 1086, Germany) for 15 min. The samples were centrifuged at 3250 rpm for 30 min and 1 mL of the supernatant was transferred to a test tube. The extract was kept in a boiling water bath for 15 min after addition of 2 mL of 0.5 mol L−1 sodium citrate buffer (pH 5.0) and 1 mL ninhidrin reagent. One millilitre of 60 % ethanol was added to the cooled mixture. The absorbance measurement was made against blank at 570 nm (Evolution 160 UV–visible). Total free amino acid content was expressed as mg glutamic acid equivalents kg−1 sample [23]. Texture analysis Texture measurements were carried out using texture analyser TA.XT2 (Stable Micro Systems, Godalming, U.K.) with 5 kg load cell. Texture profile analysis (TPA) was performed with compression test. Toughness was measured using shearing test. For TPA, the samples were compressed by cylindrical probe of texture analyser (TA.XT2). From the moment the probe in contact with the sample two sequential compressions with 5 mm/s speed were applied until reaching 40 % their original height. Hardness, springiness, gumminess, cohesiveness and chewiness were determined.
Effect of bromelain and papain enzymes addition on physicochemical and textural properties of squid…
For shearing test, blade of texture analyser (TA.XT2) was brought down onto the sample. The first test speed was 1 mm/s, the last speed was 5 mm/s. Toughness was determined by maximum force (kg) necessary to cut the sample. Sensory texture analysis Sensory analysis was conducted by a sensory panel. Six trained panellists (three females, three males) from the staff members in Fisheries Faculty conducted the panel. Panellists aged between 25 and 50 and had experience in evaluating of seafood and accustomed to consume cephalopods. Ability of panellists to detect differences in hardness was tested. Four reference foods form the standard hardness scale [24] were presented to the panellists. Panellists having ability to place the foods in true order were used in the panel of present study. Squid pieces were evaluated in terms of hardness, chewiness and elasticity attributes using ninepoint descriptive scales as follows: 1 = excellence 9 = very poor for hardness, chewiness and elasticity. The samples were placed in a jar and put into water bath at 90 °C for 30 min. After cooking the samples left for cooling to be warm. The samples were coded using letters and randomly presented to the panellists. Distilled water was provided to purge the palate of residual flavour notes between samples. All assessments took place in individual booths in a day light conditions. Statistical analysis Two replications of the experiment were conducted at separate times and all analyses were performed in duplicates. Data were analysed by a two factor factorial arrangement in a completely randomized design. The two
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factors were the two enzyme treatments (papain and bromelain) and the two concentrations (0.001 and 0.004 %). Analysis was conducted using the SAS software (Statistical Analysis System, Cary, NC, USA). Data were subjected to analysis of variance followed by Duncan’s multiple range tests.
Results and discussion Soluble protein Muscle proteins have significant roles for the structure, function, and integrity of the muscle. Proteins incur changes during the conversion of muscle to meat that affect tenderness; and additional changes occur during further processing, through the formation of peptides and free amino acids as a result of the proteolytic enzymatic activity [25]. Basically, the tenderness of meat is much linked with the toughness of meat as a consequence of interconnectedness between connective tissues and myofibrils that enclosed the muscle protein. Change in solubility under a variety of extracting conditions has been taken as a measure of change in protein conformation, i.e., denaturation, and has been used as an indicator of the quality change that a stored muscle food has undergone [26]. Types of proteins and their functional status are two factors that most influence the texture of cephalopod muscle. In this experiment total soluble protein content of squid significantly increased after treatment with enzymes (Fig. 1). Effects of type and concentration of enzyme were insignificant (p > 0.05). These increases probably occurred thanks to proteolysis by enzymes. Increases in proteolysis in chicken, fish, and beef and pork muscles after treatment with enzymes have been reported [16].
Fig. 1 Soluble protein content of squid muscle treated with bromelain and papain enzymes. Different letters above individual bars indicate significant differences between experimental groups (p
