Improvement of lycopene extraction from tomatoes by enzyme – assisted treatment - Vu Phuong Nam

Journal of Science and Technology 54 (4A) (2016) 275-282 1 IMPROVEMENT OF LYCOPENE EXTRACTION FROM TOMATOES BY ENZYME – ASSISTED TREATMENT Vu Phuong Nam 1 , Lai Quoc Phong 1, * , Truong Huong Lan 1 , Pham Thu Thuy 2 1 Food Industry Researches Institute, Ministry of Industry and Trade, 301 Nguyen Trai Road, Thanh Xuan District, Hanoi 2 School of Biotechbology and Food Technology, Hanoi University of Science and Technology, 1 Dai Co Viet Street, Hai Ba Trung District, Hanoi * Email: quocphong.lai@ensiacet.fr Received: 15 August 2016; Accepted for publication: 7 October 2016 ABSTRACT Lycopene is a natural carotenoid pigment and a high value nutraceutical having wide use in the food, feed, medicinal and cosmetic industries. Both food researchers and producers always attract enhancement of the lycopene extraction yield from tomatoes. In this work, improvement of lycopene extraction by enzyme – assisted treatment from tomatoes was studied. Hydrolytic enzymes such as cellulases and pectinases were firstly screened for enzymatic treatment of tomato purée, then, some process parameters of the enzymatic treatment of tomatoes were optimized, with respect to concentration of the enzymes, tomato purée/water ratio, pH of tomato purée, temperature and incubation time. Lycopene extraction of enzymatic treatment of tomato purée by mix of solvents (hexane, acetone and ethanol: 2:1:1v/v/v) showed that: Pectinex Ultra Clear preparation attributed significantly to increase the extractability of tomato lycopene in comparison to Viscozymes L, Cellulast 1.5 and control samples (no enzyme addition). The highest lycopene yield was obtained, reached 163.35 mg/kg at their optimum parameters of enzymatic treatment, such as tomato purée/water ratio: 1/0; enzyme addition: 1 %; pH: 5.0; temperature: 50 o C and incubation time: 60 minutes. Keywords: tomatoes, lycopene, pigment, enzyme, extraction. 1. INTRODUCTION Lycopene, a carotenoid, presents in high concentration in tomatoes and tomato products, has attracted considerable attention, as epidemiological evidence continues to suggest that it may provide protection against cancer and other degenerative diseases, influenced by free radical reaction [1]. Recent epidemiological studies revealed that the intake of tomatoes and blood lycopene level are inversely associated with the risk of developing cancers at several anatomical sites, including the prostate gland, stomach, and lung [2]. Conjugated carbon-carbon double bonds provide lycopene with its antioxidant properties, by successful delocalization of captured free radical species. Lycopene is now in high demand not only by pharmaceutical companies but Phuong Nam Vu, Quoc Phong Lai, Huong Lan Truong, Thu Thuy Pham 276 also for the food, feed, and cosmetic industries. Tomatoes and tomato products are considered as one of the best sources of lycopene. Thus, it is very important to extract and recovery lycopene form tomatoes. Tomato lycopene are usually extracted by organic solvents such as acetone, hexane, ethanol, ethyl acetate or by mix of 2 solvent hexane/acetone of hexane/ethanol Moreover, some recent studies showed that tomato lycopene could be obtained by using of ultrasound wave, microwave, high pressure extractions and enzyme assisted treatment [3]. Cell-wall degrading enzymes have been successfully used to favor the release of a variety of components, including vegetable oil, non-volatile grape aroma precursors and carotenoids from vegetable tissues. Choudhari and Ananthanarayan [3] have investigated their use for improving the extraction of lycopene from tomatoes. These authors used two enzymes, cellulose and pectinase, and found that both of them allowed a significant increase in lycopene recovery from various tomato materials. Recently, Zuorro et al. [2] have shown that the recovery of lycopene with hexane from the peel fraction of tomato processing waste can be greatly enhanced by the use of mixed enzyme preparations [3]. More recently, in the study of Ranveer et al. [5], cellulose (1.5 %) and pectinase (2 %) of Aspergillus niger at four hours of incubation period, followed by solvent extraction, proved to be advantageous for the lycopene extraction from the tomato industrial waste. The objective of the present work was to obtain a high extraction yield of lycopene from fresh tomatoes by optimizing of enzymatic treatment before extraction process by mix of solvents. 2. MATERIALS AND METHODS 2.1. Materials 2.1.1. Raw materials Fresh tomatoes of ‘Montavi’ variety of India were grown in Dong Anh district of Ha Noi, Viet Nam and were purchased from Bac Ha Company. The tomatoes were stored at 2-8 o C for a maximum of 48 hours before used. 2.2.2. Enzymes and chemicals Pectinex Ultra Clear, Cellulast 1.5L, Viscozyme L of Novozyme Denmark were purchased from Brentag Company. Pectinex Ultra Clear produced from a fungal source Aspergillus aculeatus has a declared activity of 7,900 PGNU/ml. Cellulast 1.5L produced from a fungal source Trichoderma reesei and has a declared activity cellulase of 700 U/g. Viscozyme L produced from a fungal source Aspergillus niger and has cellulolytic enzyme mixture of 100 FBG/g. The HPLC grade solvents i.e. hexane, ethanol, acetone were used for lycopene extraction, whereas butylated hydroxytoluene (BHT) of analytical grade was used for this experiment. 2.2. Methods 2.2.1. Sample preparations Whole tomatoes were washed and drained in ambient temperature for 30–60 minutes. They were crushed and blended by Phillip HR 2068 blender to produce tomato purée. Improvement of Lycopene extraction from tomatoes by enzyme-assisted treatment 277 2.2.2. Screening of enzyme preparations The three enzyme preparations were screened by their capacity of enhancement of lycopene extraction from tomatoes. Each 200 g of tomato purée was put into the reactors of 1L and then added respectively 1 % w/w of Pectinex Ultra Clear, Cellulast 1.5 L and Viscozyme L. The reaction mixture was continuously stirred at 250 rpm, at pH 4.5, at 50 o C, during 120 min. The control sample was carried out by similarly protocol but no enzyme addtion. At the end of the enzymatic treatment, samples were taken for lycopene extraction. 2.2.3. Optimization of the enzymatic treatment of tomato purée for lycopene extraction Effect of the ratio of tomato purée/water: Tomato purée was diluted by water with ratios from 1/0 to 1/3 with addition of Pectinex Ultra Clear at 1 %. The parameters of the enzymatic treatments were kept as the same ones as described in the section 2.2.2. After 120 minutes of the enzymatic treatment, samples were taken for lycopene extraction. Effect of the enzyme concentration: This experiment for lycopene extraction was carried out with different enzyme concentrations: 0.2; 0.5; 1 and 2 % (w/w of tomato purée), by using ratio of tomato purée/water of 1/0, stirring at 250 rpm, pH 4.5 and 50 o C. After 120 minutes of the experiment, samples were taken for lycopene extraction. Effect of the pH of tomato juice: Different pH of 4.0; 4.5; 5.0 and 5.5 were applied for enzymatic treatment, by using enzyme concentration of 1% w/w, ratio of tomato purée/water of 1/0, stirring at 250 rpm and 50 o C. After 120 minutes of each enzymatic treatment, samples were taken for lycopene extraction. Effect of the extraction temperature: Different temperature at 40, 45, 50 and 55 0 C were used in the enzymatic treatment, with the enzyme concentration of 1 % w/w and the ratio of purée tomato/water of 1/0, stirring at 250 rpm and pH 5.0. After 120 minutes of each enzymatic treatment, samples were taken for lycopene extraction. Effect of the incubation time: Enzymatic treament was investigated with ratio of tomato purée/water of 1/0, enzyme concentration of 1 % w/w, the temperature at 50 o C, pH 5.0, with continuously stirring at speed of 250 rpm. Then, the samples were withdrawn at times of 5; 30; 60; 90 and 120 minutes for lycopene extraction. 2.2.4. Lycopene extraction and yield calculations Lycopene extraction: At the end of each treatment, 50 ml samples were centrifuged at 6.000 rpm, 15 mins, at 5 o C to obtaine the tomato pulp for lycopene extraction. Lycopene extraction by mix of 3 solvents (hexane:acetone:alcohol (2:1:1v/v/v) was described previously by Wayne W. Fish [6]. Briefly, approximately 0.5 g sample was weighed from each tomato pulp into 100 ml flask and then added 3 mix solvents containing 5 ml of 0.05 % (w/v) butylated hydroxytoluene (BHT) in acetone, 5 ml of ethanol, and 10 ml of hexane. Sample was extracted for lycopene on an orbital shaker at constant speed 180 rpm, for 15 min. After shaking, 3 ml of deionized water was added to this flask and the samples were shaken for an additional 5 min. Then, the flask was left at room temperature for 5 min to allow for phase separation. The absorbance of the upper, hexane layer was measured in a 1 cm path length quartz cuvette at 503 nm by a double-beam UV–VIS spectrophotometer (HALO DB - 20) blanked with hexane. Phuong Nam Vu, Quoc Phong Lai, Huong Lan Truong, Thu Thuy Pham 278 Calculation of lycopene extraction yields The lycopene content of each sample in one experiment was calculated: H: Lycopene content (mg/kg pulp); OD (503) = Absorbance at the wave length 503 nm; 31,2 = coefficient; n: dilution factor; m: gram of sample. The lycopene extraction yield of each experiment was calculated: G: Lycopene extraction yield (mg/kg whole tomato); M: g of obtained pulp after centrifugation. Each experiment was triple to have the average value of lycopene extraction yield. Then, these values presented in the figures of this report were the average values. 3. RESULTS AND DISCUSSION 3.1. Screening of enzyme preparations for enzymatic treatment In the first experiments of this study, three enzyme preparations, including Pectinex Ultra Clear, Cellulase L and Viscozyme 1.5 L were screened to find out the most suitable enzyme for the pretreatment of tomatoes. The results in Fig. 1 show that all of the enzymatic treatments of tomato purée increased the lycopene extraction yield significantly in comparison to the untreated sample. This observed increase in yields can be explained by considering that pectins, celluloses and hemicelluloses are major polysaccharide components of tomato purée which could be more or less degraded by the enzymes used, thus favoring solvent penetration and lycopene dissolution from the tomato tissues. In this study, the highest lycopene extraction yield was obtained with Pectinex Ultra Clear, reached 88.05 mg/kg, followed by sample treated with Cellulast 1.5 L, reached 69.43 mg/kg. The lowest lycopene extraction yield was obtained with sample treated with Viscozyme L, reached 63.58 mg/kg. According to current views of plant cell – war architecture, cellulose, a linear polymer of β-1,4 – linked glucose, and hemicelluloses, such as xyloglucans and xylans, 59 69.43 88.05 63.58 0 50 100 150 200 Control Cellulast 1.5 L Pectinex Ultra Clear Viscozyme Enzyme preparations L y co p en e ex tr a ct io n y ie ld ( m g /k g ) Figure 1. Effect of enzyme preparations of enzymatic treatment on lycopene extraction. 98.5 93.5 89 75.5 0 50 100 150 200 1/0 1/1 1/2 1/3 Ratios of tomato purée/water L y co p en e ex tr a ct io n y ie ld ( m g /k g ) Figure 2. Effect of tomato purée/water ratio of enzymatic treatment on lycopene extraction. Improvement of Lycopene extraction from tomatoes by enzyme-assisted treatment 279 form a fairly rigid network that is embedded in, and interacts with, a gel – like matrix of hydrated pectin substances [7]. In our study, Pectinex Ultra Clear preparation has been declared as polygalacturonase enzyme, able to cleave the pectin substances in the cell walls of tomato purée more efficient than Cellulast 1.5L and Viscozyme L, which have only cellulolytic activity. Therefore, lycopene was easier extracted by solvents from the tomato purée treated by Pectinex than by the other enzymes. Our results are also coincided with the previous findings of Choudhari & Ananthanaraya [4] who reported that using pectinase for the enzymatic treatment of whole tomatoes increased the lycopene extraction more efficiency than using cellulase. 3.2. Optimization of some technical parameter of enzymatic treatment of tomato purée 3.2.1. Effect of tomato purée/water ratio of enzymatic treatment on lycopene extraction yield To choose the suitable ratio of tomato purée/water for the lycopene extraction, the experiments were carried out with some different ratios of 1:0; 1:1; 1:2 and 1:3. The lycopene extraction yield was achieved at the highest value with the ratio of tomato purée/water at 1/0 (no water addition), reached 98.5 mg/kg. When the ratio of tomato purée/water was lower, the lycopene extraction yield decreased. For ratio of tomato purée/water was 1/3, the lycopene extraction yield is the lowest, reached 75.5 mg/kg (Fig. 2). Therefore, it is better to use the tomato purée without water addition before the enzymatic treatment of tomato purée for lycopene extraction. 3.2.2. Effect of enzyme concentration of enzymatic treatment on the lycopene extraction Enzyme reactions are reversible and depend on many factors, including the enzyme concentration which affects the ability of the cleavage of substrates. To optimize the enzyme concentration of Pectinex Ultra Clear, the experiments were carried out with different enzyme concentrations of 0.2 %; 0.5 %; 1 % and 2 %. Figure 3 shows the lycopene extraction yield was the highest by the addition of enzyme at 1 %, reached 115.02 mg/kg, while for the addition 0.2 %, the lycopene extraction yield reached the lowest level of 55.6 mg/kg. However, when the enzyme concentration increased to 2 % w/w, the lycopene extraction yield is reduced, only 101.2 mg/kg. Our obtained results were not similar with those of some previous reports. For example, Choudhari and Anathanarayan [4] showed that the optimum pectinase concentration was 0.5 % to digest the whole tomatoes [4], while Ranveer et al. [5] reported that the recovery efficiency of 55.6 97 115.02 101.4 0 50 100 150 200 0.2% 0.5% 1% 2% Enzyme concentration L y co p en e ex tr a ct io n y ie ld ( m g /k g ) Figure 3. Effect of enzyme concentration of enzymatic treatment on the lycopene extraction. 113.15 102.43 133.25 103.17 0 50 100 150 200 4.0 4.5 5.0 5.5 pH L y co p en e ex tr a ct io n y ie ld ( m g /k g ) Figure 4. Effect of pH of the enzymatic treatment on the lycopene extraction. Phuong Nam Vu, Quoc Phong Lai, Huong Lan Truong, Thu Thuy Pham 280 lycopene from tomato wastes was the highest with pectinase concentration of 2 %. These differences would be taken into account of raw materials and the activity of enzymes. 3.2.3. Effect of pH of enzymatic treatment on the lycopene extraction Enzymes are affected by changes of pH. The most favorable pH value is the point where the enzyme is most active, known as the optimum pH. To find out the optimum pH for enzymatic treatment, experiments were carried out at different pH of 4.0; 4.5; 5.0 and 5.5. Figure 4 shows that pH influenced significantly to the lycopene extraction yield from tomato purée. At pH 5.0, the highest lycopene extraction yield is 135.25 mg/kg, the lowest value was found at pH 4.0, only reached 102.43 mg/kg. This can be explained high or low pH values generally result in some reductions of activity for most enzymes. However, Papaioannnoua et al. [8] showed that there were no significant influence of pH on the extraction yields of lycopene from tomato peels with the enzymatic treatment by two enzyme preparations Citrozyme Ultra L and Citrozyme CEO. 3.2.4. Effect of temperature of enzymatic treatment on lycopene extraction Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases when the temperature raise. Variations in reaction temperature as small as 2 or 5 degrees may introduce changes of 10 to 20 % in the results [9]. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. In order to find out the optimum temperature for the enzymatic treatment, some experiments were carried out at different temperatures of 40 o C, 45 o C, 50 o C and 55 o C. The results in Fig. 5 show that lycopene extraction yield reached the highest at 50 o C (135.39 mg/kg), while the lowest lycopene lycopene extraction yield was observed at 40°C (68.74 mg/kg). When the temperature heated to 55 °C, lycopene extraction yield decreased significantly, reached only 75.75 mg/kg. This can be explained in high temperature, enzyme was partially inactivated, while in the low temperature, enzyme has not been activated, thus it leads to lower lycopene recovery efficiency. 3.2.5. Effect of incubation time of enzymatic treatment on lycopene extraction To choose suitable incubation time for the enzymatic treatment of tomato purée, the lycopene extraction yields were determined at different incubation times of 5 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes. Figure 6 shows that lycopene extraction yield was the highest after 60 minutes, reached 163.35 mg/kg. Increasing the incubation time over 60 68.74 103.28 135.39 75.75 0 50 100 150 200 40°C 45°C 50°C 55°C Temperature L y co p en e ex tr a ct io n y ie ld ( m g /k g ) Figure 5. Effect of temperature of enzymatic treatment on the lycopene extraction. 64.33 96.67 142.89 138.43 163.35 0 50 100 150 200 5 mins 30 mins 60 mins 90 mins 120 mins Incubation times L y co p en e ex tr a ct io n y ie ld ( m g /k g ) Figure 6. Effect of incubation time of enzymatic treatment on lycopene extraction. Improvement of Lycopene extraction from tomatoes by enzyme-assisted treatment 281 minutes resulted in progressive reduction in yields of lycopene extraction. In fact, after 90 minutes of the enzymatic treatment, lycopene extraction yield decreased to 142.89 mg/kg and after 120 minutes of the enzymatic treatment, it reached 138.43 mg/kg. This suggested that the enzymatic degradation of cell-wall components is very fast and occurs within the first hour of incubation. Therefore, the vast majority of lycopene molecules contained in the plant tissue is likely to be rapidly released from the protective chromoplast structures and exposed to the conditions of the external environment. Because of their high reactivity, the released lycopene molecules can undergo rapid oxidative degradation [10]. Although the underlying mechanisms are only partially elucidated, it has been shown that lycopene oxidation leads to the formation of several cleavage products, including apo-lycopenals/ones and apo-carotendials, whose spectra are shifted to shorter wavelengths compared to that of lycopene [11]. Therefore, a decrease in the lycopene content with increasing incubation time may be due to oxidation of lycopene [4]. Our result is similar to the finding in the study of Lavecchia and Zuorro (2008) who published the optimum extraction conditions treated by enzyme (Peclyve LT) within 1 hour [12]. 4. CONCLUSIONS The results of this study indicated that the lycopene extraction form tomatoes can be greatly enhanced by pretreatment of tomato purée by pectinase (Pectinex Ultra Clear) and cellulase enzymes (Cellulas 1.5L and Viscozyme L). The optimum conditions for enzymatic treatment of tomato purée by Pectinex Ultra Clear for lycopene extraction were determined, such as tomato purée/water ration: 1/0; enzyme addition: 1 % w/w; pH: 5.0; temperature: 50 0 C and incubation time: 60 minutes, thus resulted the highest lycopene yield, upto 163.35 mg/kg. Overall, the above points strongly supported the possibility of using commercial enzyme preparations to obtain lycopene from tomatoes. Acknowledgements. Thank to Ministry of Industry and Trade of Vietnam for the financial support of this study. REFERENCES 1. 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Lavecchia R., and Zuorro A. - Improved lycopene extraction from tomato peels using cell- wall degrading enzymes, European Food Research and Technology 228 (1) (2008) 153- 158. TÓM TẮT NÂNG CAO HIỆU SUẤT CHIẾT TÁCH LYCOPENE TỪ CÀ CHUA BẰNG PHƢƠNG PHÁP TRÍCH LY CÓ SỰ HỖ TRỢ CỦA ENZYME Vũ Phƣơng Nam 1, Lại Quốc Phong 1, *, Trƣơng Hƣơng Lan 1 và Phạm Thu Thủy 2 1Viện công nghiệp thực phẩm, Bộ Công thương, 301 Nguyễn Trãi, Thanh Xuân, Hà Nội 2Viện Công nghệ Sinh học và Thực phẩm, Trường Đại Học Bách Khoa Hà Nội, 1 Đại Cồ Việt, Hai Bà Trưng, Hà Nội * Email: quocphong.lai@ensiacet.fr Lycopene là một hợp chất carotenoid mầu đỏ tự nhiên và có giá trị y dƣợc cao, đƣợc sử dụng rộng rãi trong các ngành công nghiệp thực phẩm, thức ăn chăn nuôi, dƣợc phẩm và mĩ phẩm. Hiện nay, việc nâng cao hiệu suất chiết tách lycopene từ cà chua đang đƣợc nhiều nhà nghiên cứu và sản xuất quan tâm chú ý. Trong nghiên cứu này, việc nâng cao hiệu suất chiết tách lycopene từ cà chua bằng phƣơng pháp trích li có sự hỗ trợ của enzyme đã đƣợc thực hiện. Ban đầu, các enzyme thủy phân xenlulase hoặc pectinase đƣợc lựa chọn cho quá trình xử lí purée cà chua. Tiếp theo, một số thông số công nghệ của quá trình xử lí puree cà chua bằng Pectinex Ultra Clear đã đƣợc tối ƣu. Kết quả trích li lycopene bằng hỗn hợp dung môi (hexane, acetone and ethanol: 2:1:1 v/v/v) từ purée cà chua đã đƣợc xử lí enzyme cho thấy chế phẩm enzyme Pectinex Ultra Clear đã làm tăng đáng kể khả năng trích li lycopene so với các chế phẩm enzyme khác là Cellulast 1.5 L và Viscozymes L, và đặc biệt là so với mẫu đối chứng không sử dụng enzyme. Quá trình thủy phân bằng enzyme Pectinex Ultra Clear đã đƣợc tối ƣu ở điều kiện: tỉ lệ nguyên liệu/nƣớc là 1/0; nồng độ enzyme là 1 % w/w; pH 5,0; nhiệt độ 50 oC và thời gian 60 phút với hàm lƣợng lycopene đƣợc trích li từ cà chua là 163,35 mg/kg. Từ khóa: cà chua, lycopene, chất mầu đỏ, enzyme, trích li.