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Journal of Science and Technology 54 (4A ) (2016) 55-62 EFFECT OF ULTRASOUND ON PRETREATMENT OF TUNA SKIN FOR GELATIN PRODUCTION Đang Minh Nhat1, *, Chau Thanh Hien2 1 The University of Da Nang-University of Science and Technology, 54 Nguyen Luong Bang,Lien Chieu, Da Nang 2 Da Nang College of Food Industry, 101B Le Huu Trac, Da Nang * Email: dangminhnhat@dut.udn.vn Received: 15 August 2016, Acceptedfor publication: 5 October 2016 ABSTRACT This paper presents the result of study on the effect of ultrasound on extraction yield and quality of gelatin obtained from tuna skins using alkaline treatment method and alkaline-acid treatment method with ultrasound assistance. The results showed that ultrasound could shorten the time of alkaline treatment, while ensured the yield and the quality of obtained gelatin. Accordingly, the appropriate parameters of ultrasound mode that gave the best yield and quality of gelatin were determined as follows: for alkaline treatment method: amplitude of 80 %, period of 0.8s and ultrasound time of 90 min; for the alkaline-acid treatment method: amplitude of 8 0%, period of 0.8s and ultrasound time of 30 min. The former method produced gelatin with better yield and gel strength compared to the latter one (22.7 % and 143.3 g in comparison with 15.8 % và 122.4 g, respectively). The method with ultrasound support gave the gelatin with better quality in term of lipid content and gel strength in comparison with the traditional alkaline treatment method without ultrasound assistance. Keywords: fish gelatin, ultrasound, tuna, alkaline treatment, alkaline-acid treatment. 1. INTRODUCTION Extraction of gelatin from fish skins for applying in food, pharmaceutical industries, medicine, cosmetics and photography have attracted greater interest recently. Various works have shown that the extraction of gelatin from fish skins would achieve high yield and good quality when fish skins are pretreated with lime or combination of lime and acid. However, the disadvantage of these methods is long time of the pretreatment, 5 days may be needed [1, 2, 3]. Thus, finding some way to reduce time of the fish skin pretreatment with lime is necessary. Dang Minh Nhat, Chau Thanh Hien Meanwhile, ultrasound, when propagates in liquid media, releases a great amount of energy which could cause significant mechanical impact.Therefore, if the ultrasound during the pretreatment of fish skin was applied, the change of gelatin extraction yield would be expected.There was a work concerning the application of ultrasound in gelatin production from fish for the extraction step but not for pretreatment step [4]. So, the aim of our work was to evaluate the possibility of using ultrasound in tuna skin pretreatment with lime in order to shorten the time of pretreatment. 2. MATERIALS AND METHODS 2.1. Materials, chemicals Tuna skins were obtained from seafood processing companyBắcĐẩu, Da Nang City. Chemicals:Ca(OH)2, CH3COOH of analytical grade. 2.2. Methods 2.2.1.Determination of moisture and ash contentof tuna skins: according to methods of AOAC 952.08 and AOAC 938.08 (1990) [5]. 2.2.2. Determination of gelatin solution viscosity after extraction:Using viscometerBrookfield LVDEat 60 oC. 2.2.3. Calculation of extraction yield [3]:Extraction yield is expressed as percentage of the weight of obtained gelatin (dried to 11 % moisture) to the weight of raw material of fish skin. 2.2.4. Determination of crude protein content ofgelatin: according to the method ofTCVN 3705:1990. 2.2.5.Determination of lipid content of gelatin: according to the method of TCVN 3703:2009. 2.2.6. Measurement of gel strength of gelatin Prepare a gelatin solution of 6.67 % at ambient temperature, heat and hold at 65 oC for 25 min. Pour the solution into PE bags of 3 cm diameter. Let it cool down at room temperature for 15 min and later to 10 oC in refrigerator for 16÷18 h. Gel strength was measured by Rheo Tex (piston diameter of 12.7 mm, penetration velocity 0.5 mm/s, at 10 oC) [3]. 2.2.7. Data processing methods The results of experiments were expressed as the average of triplicates. Significant difference tests were carried out using Minitab 16 at significant level α of 5 %. 56 Effect of ultrasound on pretreatment of tuna skin for gelatin production 2.3. The procedure of gelatin extraction from tuna skin with ultrasound assistance - Fish skins obtained from plant were washed with chlorine 50 ppm, stored at -20 ÷ -30 oC. Before using for experiment, the skin materials were unfrozen, washed, cut into pieces of 2 ÷ 3 cm2, squeezed and thoroughly mixed. Samples of 50 g were used for each experiment. - Samples were exhibited to ultra sound in the LABSONICR P(Germany) while submerged in Ca(OH)2 20 g/l at the ratio of skin/solution of 1/6 (w/v) at 20 oC. - After the application of ultrasound, samples were washed several times with clean water to achieve the neutral pH. They were then extracted with distilled water at the ratio of skin/water of 1/3 (w/v) at 60 oC. The extract solutions were filtered and measured for the viscosity. The solutions were also poured into trays and dried at 45 oC to the end moisture of 10÷12 %. Dried films of gelatin were weight to determine the extraction yield. - For the pretreatment method of combination of lime and acid, samples were also processed in the same way. However, after the application of ultrasound, the materials were washed and submerged in acid acetic 7.5 mM before extraction and drying [1, 4]. 3. RESULTS AND DISCUSION 3.1. Effect of ultrasound on tuna skin pretreatment with lime In this study, we investigated the ultrasound parameters including amplitude, period and time of application. The effectiveness of fish skin pretreatment with ultrasound assistance was evaluated by the amount of obtained gelatin (extraction yield) and the quality of gelatin, including viscosity and gel strength. 3.1.1. Effect of ultrasound amplitude on quality and extraction yield of gelatin The ultrasound period and time of ultrasound application were set at 0.8 s and 90 min, respectively and the effect of ultrasound amplitude was examined at level: 50 %, 60 %, 70 %, 80 %, 90 % and 100 % [4]. Experiments were carried out as described in the section 2.3 and the results of these experiments are shown in Figure 1. Figure 1 showed that amplitude had great effect on the viscosity of the extract solution and the gel strength of obtained gelatin, meanwhile the extraction yield was less affected. In general, all viscosity, extraction yield and gel strength increased gradually with the increase of amplitude from 50 % to 80 % and then started to decrease. At the amplitude of 80 %, viscosity, yield and gel strength reached the maximum value of 14.7 cP, 20.1 % and 143.3 gram, respectively. This could be explained by the fact that at different amplitude level, the released energy affecting samples was different. At low amplitude, the released energy was not strong enough to break the structure of fish skin and the cross-links in collagen structure, thus polypeptide chains of collagen were stuck in skin structure and could not be extracted to extracting water. On the other hand, when the amplitude was too high, besides its effect of breaking cross-links of collagen and releasing non-collagen components from the skin, it could also cut the polypeptide chains of collagen into small fragments 57 Dang Minh Nhat, Chau Thanh Hien 40 35 30 25 20 15 10 5 0 133b 127.1c 124.5e 102.2f 20.1ab 20b 14.7a 13.3d 13.2d 12.6e 120 20.3a 20.1ab 150 126d 143.3a 20.1ab 13.8b 90 19.7c 13.5c 60 30 Gel strength, gram Viscosity, cP; Yield, % which could be lost during washing step, leading to the lower extraction yield, lower viscosity and lower gel strength of obtained gelatin [4]. 0 50 60 70 80 Amplitude, % Viscosity, cP 90 Yield, % 100 Gel strength, gram Figure 1. Effect of ultrasound amplitude on viscosity, extraction yield and gel strength of gelatin. 3.1.2. Effect of ultrasound period on quality and extraction yield of gelatin 40 35 30 25 20 15 10 5 0 121.3a 95.4e 101.3d 13.4a 10.1d 22.6a 22.1b 21.4d 114.2c 115.7b 11.2c 120 100 21.7c 17.6e 13.5a 140 80 60 11.4b 40 20 0.6 0.7 Viscosity, cP 0.8 Yield, % 0.9 Gel strength, gram Viscosity, cP; Yield, % The ultrasound period of the equipment is defined as the time (seconds) of emitting ultrasound in one second. In this study, we set ultrasound amplitude, time of application at 80 % and 90 min respectively, and investigated ultrasound period at level 0.6, 0.7, 0.8, 0.9 and 1. The effect of period on the quality and extraction yield of gelatin is shown in Figure 2. 0 1 Period, s Gel strength, gram Figure 2. Effect of ultrasound period on viscosity, extraction yield and gel strength of gelatin. From Figure 2, it could be seen that when the period was prolonged from 0.6s to 0.8s, all viscosity, extraction yield and gel strength of extracted gelatin increased. The maximum values of 58 Effect of ultrasound on pretreatment of tuna skin for gelatin production viscosity, extraction yield and gel strength were 13.5cP, 22.6 % and 121.3 gram at the period of 0.8s. But they all decreased as the period was increased furtherto 1s. At short period the mechanical impact of ultrasound was not strong enough to alter the skin and collagen structure, so few gelatin molecules were extracted. On the other hand, if period was too long, the ultrasound could cause the polypeptide chains to fragment and wash away. Thus, the final extraction yield decreased. The viscosity and the gel strength were also lower as the consequence of fragmentation of collagen chains. 3.1.3. Effect ofultrasoundtime on quality and extraction yield of gelatin In this study, the amplitude and the period were set at 80 % and 0.8 s respectively and the effect of application time was investigated at 60 min, 75 min, 90 min, 105 min and 120 min. Experiments were carried out as described in section 2.3 and the results were expressed in Figure 3. 35 30 25 20 15 140 122.6a 102.1b 97.3c 89.3d 22.4b 11.7c 22.1c 12.6b 22.7a 13.5a 120 86.4 21.7d 22.4b 80 60 13.6a 9.3d 10 100 40 Gel strength, gram Viscosity, cP; Yield, % 40 20 5 0 0 60 75 90 105 120 Ultrasound time, min Viscosity, cP Yield, % Gel strength, gram Figure 3. Effect of ultrasound time on extraction yield, viscosity and gel strength. The result from Figure 3 showed that as the ultrasound application prolonged from 60 min to 90 min, extraction yield, viscosity and gel strength of obtained gelatin had increasing tendency, but if ultrasound was further applied after 90 min, they all started to decrease. The optimum of application time therefore was 90 min, at which the yield and gel strength reached maximum values of 22.7 % and 122.6 gram respectively. Meanwhile, the viscosities measured at 90 min and 105 min were not significantly different. The explaination for these courses of extraction yield, viscosity and gel strength could be similar to what were described previously, i.e. due to change in fish skin and collagen structure as a consequence of the impact of ultrasound. To conclude, the best conditions of ultrasound application for tuna skin pretreatment in lime were amplitude 80 %, period 0.8 s and application time 90 min. 59 Dang Minh Nhat, Chau Thanh Hien 3.2. Effect of ultrasound on tuna skin pretreatment using combination of lime and acid The results of some works have shown that high extraction yield and quality of gelatin from tuna skin could be achieved by using combination of lime and acid for pretreatment, accordingly the skin was submerged in lime 2 days and then in acetic acid for 3 hours [1, 3]. Because of the short time of acid pretreatment, in this study we aimed to use ultrasound for reduction of the pretreatment time with lime.We chose amplitude of 80 %, period of 0.8 s and examined the effect of time of ultrasound application on skin pretreatment with lime at various levels: 20 min, 25 min, 30 min and 35 min. Samples after pretreatment with lime were washed with water to neutral pH and submerged in acetic acid 7.5 mM for 60 min before extraction and drying as described in section 2.3. The results were shown in Figure 4. 25 110.2d 121.2b 140 120 100 20 15 122.4a 114.3c 13.1a 14.6c 15.5b 12.8b 15.4b 15.8a 13.2a 12.8b 10 80 60 40 5 Gel strengh, gram Viscosity, cP; Yield, % 30 20 0 0 20 Viscosity, cP 25 30 Ultrasound time, min Yield, % 35 Gel strength, gram Figure 4. Effect of ultrasound time on viscosity, extraction yield and gel strength. Figure 4 indicated that viscosity, extraction yield and gel strength were directly proportional to time of ultrasound application as it increased from 20 min to 30 min.The maximum of viscosity, yield and gel strength reached at 30 min was 13.2 cP, 15.8 % and 135 gram, respectively. If ultrasound was further applied longer than 30 min, all viscosity, extraction yield and gel strength of gelatin decreased. The results also showed that the pretreatment method of using combination of lime and acid produced gelatin with lower extraction yield, viscosity and gel strength than the method of using only lime. It could be explained that after the application of ultrasound in lime solution, fish skin became swell, its structure wasloose. So acetic acid could easily attack peptidebonds, fragmented collagen into small chains that went lost during washing step, causing the lowextraction yield and low gel strength of gelatin. 3.3. Characteristics of gelatin obtained from method of using pretreatment with lime and ultrasound assistance The aim of this study was to compare the chemical composition and quality of gelatins obtained from new pretreatment method and “traditional” method of using lime without ultrasound 60 Effect of ultrasound on pretreatment of tuna skin for gelatin production assistance. The quality of gelatin was evaluated mainly by gel strength, which was dependent on the composition of amino acids, the proportion of high molecular weight chains. Gelatin with higher gel strength is considered to be of better quality [6]. Using the traditional method of pretreatment with lime (20g Ca(OH)2/l) in 5 days and new method of pretreatment with lime (20g Ca(OH)2/l) and ultrasound assistance (amplitude 80%, period 0.8s) in 90 min, two samples were obtained and subjected to analysis. The results were shown in Table 1. Table 1. Characteristics of gelatin samples obtained from “traditional” method and new method. New method with “Traditional” method without Characteristics ultrasound assistance ultrasound assistance Crude protein, % 85.70 ± 1.03 85.71 ± 1.02 Lipid, % 0.40 ± 0.09 1.81 ± 0.12 Ash content, % 0.52 ± 0.07 0.86 ± 0.08 Moisture, % 10.25 ± 0.14 10.56 ± 0.13 Gel strength, gram 143.3 ± 1.04 106.0 ± 1.12 Viscosity, cP 14.6 ± 0.12 14.7 ± 0.13 Method Table 1 showed the difference in some characteristics of two samples produced from two methods, especially in lipid content and gel strength. The traditional method using lime for pretreatment gave gelatin with rather high lipid content in comparison with the new method also using lime for pretreatment but with ultrasound assistance (1.81 % compared to 0.4 %). Besides,the gel strength of gelatin sample from the “traditional” method was significantly lower than the new method (106.0 g compared to 143.3 g). The reason could be that the ultrasound not only helped to extract gelatin well but also homogenized lipid in skin, enabled it to be washed away from skin better during washing step. So the obtained gelatin was purer. On the other hand, shorter time of treatment with lime in new method might help the polypeptide chains of gelatin being less fragmented, thus the gelling ability was higher. 4. CONCLUSIONS Ultrasound could significantly shorten the time of tuna skin pretreatment with lime while ensuring the extraction yield and the quality of obtained gelatin. The method using only lime produced gelatin with better extraction yield, viscosity and gel strength in comparison with the method using combination of lime and acetic acid. The method of using lime and ultrasound for pretreatment of skin has showed to be suitable for obtaining gelatin from tuna skin. 61 Dang Minh Nhat, Chau Thanh Hien REFERENCE 1. 2. Châu T. H. et al. – Nghiên cứu một số yếu tố ảnh hƣởng đến hiệu suất và chất lƣợng gelatin thu nhận từ da cá ngừ đại dƣơng, Tạp chí Khoa học và Công nghệ 4B (2015) 120-125. Haiying L.et al.- Rheological properties of channel catfish (Ictaluruspunctaus) gelatin from fish skins preserved by different methods, ScienceDirect41 (2008) 1425-1430. 3. Jitender K. J. et al. - Characterization of fish gelatin from Blackspotted Croaker (Protonibeadiacanthus), Archives of Applied Science Research 4 (3) (2012) 1353-1358. 4. Hyun K. K. et al. - Effects of ultrasound treatment on collagen extraction from skins of the sea bass Lateolabrax japonicas, Food Science and Technology 78 (2012), 485-490. 5. Helrich K. - Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC), 1990. Reinhard Schrieber and Herbert G. - Gelatine Handbook, 2007, pp. 57-59. 6. TÓM TẮT ẢNH HƢỞNG CỦA SÓNG SIÊU ÂM ĐẾN QUÁ TRÌNH XỬ LÍ DA CÁ NGỪ ĐẠI DƢƠNG ĐỂ SẢN XUẤT GELATIN Đặng Minh Nhật1, *, Châu Thành Hiền2 Trường Đại học Bách khoa, Đại học Đà Nẵng, 54 Nguyễn Lương Bằng, Đà Nẵng 1 Trường Cao đẳng Lương thực Thực phẩm, 101B Lê Hữu Trác, Đà Nẵng 2 * Email: dangminhnhat@dut.udn.vn Bài báo trình bày kết quả nghiên cứu ảnh hƣởng của sóng siêu âm đến hiệu suất và chất lƣợng gelatin thu nhận từ da cá Ngừ Đại Dƣơng bằng phƣơng pháp kiềm và phƣơng pháp kiềm - acid có sự hỗ trợ của sóng siêu âm. Kết quả nghiên cứu đã cho thấy sóng siêu âm có khả năng rút ngắn thời gian ngâm vôi nhƣng vẫn đảm bảo hiệu suất và chất lƣợng gelatin thu đƣợc. Theo đó, các thông số kĩ thuật của quá trình siêu âm cho hiệu suất cao và chất lƣợng tốt nhất đƣợc xác định nhƣ sau: Với phƣơng pháp chỉ ngâm vôi, biên độ sóng siêu âm 80 %; chu kì đóng ngắt 0,8 giây và thời gian siêu âm 90 phút; Với phƣơng pháp kết hợp ngâm vôi và acid, biên độ sóng 80 %; chu kì đóng ngắt 0,8 giây, thời gian siêu âm 30 phút. Phƣơng pháp siêu âm trong dung dịch vôi đã chứng tỏ cho hiệu suất thu hồi và độ bền gel gelatin cao hơn phƣơng pháp siêu âm trong dung dịch vôi và sau đó ngâm trong dung dịch acid acetic (22,7 % và 143,3 g so với 15,8 % và 122,4 g). Phƣơng pháp siêu âm cho gelatin có chất lƣợng về hàm lƣợng lipid và độ bền gel tốt hơn so với phƣơng pháp xử lí kiểm truyền thống không có siêu âm hỗ trợ. Từ khóa: gelatin cá, sóng siêu âm, da cá Ngừ Đại Dƣơng, xử lí với vôi, xử lí kết hợp vôi-axit. 62