Study on treatment technology of “Tao Meo” using pectinase enzyme in “Tao Meo” vinegar production by submerged method

Journal of Science and Technology 54 (4A) (2016) 298-306 STUDY ON TREATMENT TECHNOLOGY OF “TAO MEO” USING PECTINASE ENZYME IN “TAO MEO” VINEGAR PRODUCTION BY SUBMERGED METHOD Nguyen Thi Viet Anh * , Bui Thi Thuy Ha Food Industries Research Institute, 301Nguyen Trai Road, Thanh Xuan, Hanoi * Email: vietanhjp27@yahoo.com Received: 15 August 2016; Accepted for publication: 7 October 2016 ABSTRACT “Táo mèo” vinegar, a fat-burner famous for its many benefits for the digestive and immune systems, can be consumed as functional drinks. However, “táo mèo” vinegar is still producing mainly in manual-based, small-scaled with the surface fermentation method and containing many hazards. For industrial scales, the application of pectinase during the fruit extraction process has increased the quality of end-products and extraction efficiency, leaving no residue during storage. This research focus on the simultaneous application of two kinds of pectinase: PectinexUltra SP-L and PectinaseUltra Clear, during the extracted processing of “táo mèo”. The results indicated that simultaneously using both enzymes has increased the extractivity of the process to 25 %, and also has increased the amounts of function substances such as vitamin C, polyphenol, sugar, acid, and soluble pectin, compared to not using enzymes. The extractive process using the two enzymes for the fruit extracts is determined as follows: PectinexUltra SP- L is used at concentration of 0.15 %, temperature is 30 o C for 60 minutes; followed by using PectinaseUltra Clear with concentration of 0.1 %, temperature is 55 o C for 60 minutes. The enzyme processing does not interfere with the vinegar fermentation process; vinegar with enzyme pretreatment contains higher amounts of polyphenol, vitamin C, and potassium compared to that without enzyme pretreatment. The amount of soluble pectin in vinegar with enzyme pretreatment stays constant with no sediment during six months of storage, whereas vinegar without enzymes pretreatment produces precipitation afer two months of storage. Keywords: “Tao meo” vinegar, pectinase, polyphenol, functional drinks. 1. INTRODUCTION “Táo mèo” (Docynia indica) is widely distributed among the mountainous areas of North Vietnam [1, 2]. The chemical compositions of “táo mèo” include organic acids, vitamin C, fibre, tanin, polyphenol, minerals...; thus its many benefits for the digestive, circulatory, and immune systems [3, 4]. “Táo mèo” vinegar is becoming more popular as a type of functional drink in the market. Besides utilizing the benefits of the “táo mèo” fruit, “táo mèo” vinegar serves to balance the acidic system in the intestines, stimulate the growth of beneficial bacteria Study on treatment technology of “Tao meo” using pectinase enzyme 299 in the digestive system, and burn fat. However, the current manufacturing of “táo mèo” vinegar is still manual-based, small-scaled, utilizing mainly the surface fermentation method; thus the slow fermentation speed and low extractivity, which, in turn, cause the low and unstable quality of end products alongside various potential hazards [2]. In order to manufacture high-quality “táo mèo” vinegar oriented to industrial scale-up with increased productivity, a number of measurements could be applied such as: use of enzyme during extraction process, conversion to the submerged fermentation method using the acetic bacteria stater; standardization of technical conditions; application of new filtering, storage technologies [5, 6]. During the vinegar processing from the fruit, the fruits are usually crushed, extracted for liquid extracts, and then fermented through alcohol and vinegar [3]. For hard fruits (such as “táo mèo”), or fruits whose original composition contains high amounts of pectin, the normal method of processing fruits yields only low extractivity, the produced vinegar usually looking opaque and containing sediment. To reduce the precipitation left by the product, as well as to increase the extractivity of the fruit, pectinase is used during the fruit processing stage. This enzyme processing stage assists in increasing the extractivity of the fruit, speeding up the fermentation process, reducing the viscosity of the extracts, facilitating the filtering process, and yielding end products leaving little or no sediment [7, 8]. The contents of this article revolve around the development of the “táo mèo” fruit processing stage using pectinase, as well as evaluate the impact of this enzyme processing on the quality of the end products in the manufacturing of “táo mèo” vinegar. Enzyme PectinexUltra SP-L and PectinaseUltra Clear (Novozyme) have been used in the extracting fruit processing stage. The fruit extracts then undergo fermentation through alcohol and vinegar, finally yielding the finished “táo mèo” vinegar containing the maximum amounts of polyphenol, vitamin C, potassium, and acetic acid. 2. MATERIALS AND METHODS 2.1. Materials „Táo mèo‟, bought in Son La, were fresh and neither decayed nor rotten. They were classified, and washed for experiment immediately after transported. Strains Saccharomyces cerevisiae 7012 and Acetobacter pomorum M5 were provided from the Collection of Industrial microorganism strain, Food Industries Research Institute. Commercial enzyme (Novozyme, Denmark): PectinexUltra SP-L (activity of polygalacturonase enzyme (PGNU/ml) > 3800) and PectinaseUltra Clear (activity of PGNU/ml > 7900). 2.2. Methods 2.2.1. Technology methods a. Raw material treatment processing by pectinase: “Táo mèo” were classified, washed and soaked in NaCl 3 % for 3 h. Clean fruits were crushed , supplemented with water (fruit/water was 1:2), mixed with PectinexUltra SP-L in suitable ratio, temperature and reaction time. Enzyme activity was inactivated at 75 o C for 5 min. After treatment with Pectinex Ultra Sp-L, the fruits then added PectinaseUltra Clear enzyme (for deeper cleavage of remained pectin Nguyen Thi Viet Anh, Bui Thi Thuy Ha 300 branch in liquid to clarify fruit liquid) with dilution ratio, enzyme ratio, temperature and reaction time consistent with experiment requirement. Enzyme activity was inactivated at 85 o C for 5 min b. Alcohol fermentation: Enzyme-extract juice was added with sugar (6 – 8 %). Yeast strain Saccharomyces cerevisiae 7012 was used for this processing, fermentation was conducted at 28- 30 o C for 5 days. After that, wine fermentation broth was filtrated. Mixture of alcohol liquid was added mineral salt, nitrogenous substances, then sterilized at 100 o C for 20 min. c. Acetic acid fermentation: After sterilization, alcohol solution was cooled, adjusted to 6 % of alcohol and 0.5 % of total acid contents. Acetobacter pomorum M5 (about 10 8 cfu/ml) stater was used and cultured at 30 o C, under saturated sterile aeration mode. Fermented solution was sterilized at 120 o C for 5 min, then stored, filtered and packaged. 2.2.2. Physiochemical analysis methods - Quantitative analysis of total acid (% w/w acetic acid): acid-base titration method [2]. - Quantitative analysis of alcohol (% v/v): solution boiling temperature measurement method on Salleron Dujardin Paris device [2]. - Quantitative analysis of total sugar: using DNS method [2]. - Quantitative analysis of pectin: based on reaction between galacturonic acid and cacbazol in sulfuric acid environment produced 5- cacboxyfucfurol which haspink purple color, maximum absorbance at wavelength of λ = 525 nm [2]. - Quantitative analysis of total polyphenol: using method in ISO 145021-2005 - Quantitative analysis of vitamin C: using iodine method [2]. - Quantitative analysis of Potassium: analyzed following TCVN 8908:2011 2.2.3. Extract efficiency: Extract efficiency (%) was calculated as follow 3. RESULTS AND DISCUSSION 3.1. Effect of pectin hydrolysis procedure on “Tao meo” extracted solution quality Pectin hydrolysis enzyme has been used in wine production technology, in order to increase efficiency of fruit extraction, easy to filter, increase juice quality as well ability of color extraction. For “tao meo”, hard fruit which is usually used in wine production or “tao meo” drink, application of hydrolysis enzyme for fruit treatment has not yet published. In order to evaluate application ability of enzyme to fruit juice quality, „tao meo” juice quality of samples extracted not using enzyme, using PectinexUltra SP-L enzyme or both PectinexUltra SP-L and Pectinase Ultra Clear enzymes was evaluated. Component of juice samples as well extract efficiency are shown in Table 1. It can be seen that treatment by pectinase leaded to increase in dissolved component such as sugar, vitamin C, pectin, polyphenol, or color intensity in comparison to the sample without enzyme treatment (control sample). Using both kind of enzyme increased extract efficiency 25% Study on treatment technology of “Tao meo” using pectinase enzyme 301 higher than control sample. Color intensity of sample which used enzyme was higher than of control sample that make product have better. Enzyme-treated juice was filtered to measure extract efficiency. We realized that enzyme-treated samples had easy-deposited grain, less suspension component than control sample. Especially, sample treated with both enzymes was filtered more easily and gave highest volume of juice. Thus, using both hydrolysis enzymes gave highest efficiency of “tao meo” juice quality as well extract during “tao meo” processing. Table 1. Effect of treatment procedure of pectinase on extracted “tao meo” juice. Treatment methods Quality of extracted juice Total sugar (g/l) Total acid (g/l) Polyphenol (g/l) Vitamin C (g/l) Pectin (%) Color intensity Extract efficiency (%) Without enzyme 12.8 5.1 2.0 0.4 0.152 0.180 100 Ultra SP-L 14.3 5.2 2.7 0.6 0.189 0.219 122 Ultra SP-L + Ultra Clear 15.7 5.2 3.0 0.7 0.223 0.295 125 To extract useful substrate as much as possible, experiment on suitable time for filtration to remove “Tao meo” waste was tested at certain periods: after enzyme treatment, after alcohol fermentation, after acetic acid fermentation. Alcohol and acetic fermentation was executed as described in part 2.2.1. Results of the vinegar quality of each period are shown in Table 2. Table 2. Effect of time to remove “tao meo” waste on vinegar quality. Production method Quality of “tao meo” vinegar Alcohol (%v/v) Total acid (g/l) Polyphenol (g/l) Vitamin C (g/l) Sugar (g/l) Color intensity After enzyme treatment 0.4 37 2.8 0.67 3.0 0.27 After alcohol fermentation 0.3 38 3.3 0.80 4.4 0.35 After acetic fermentation 0.35 32 3.2 0.79 4.3 0.36 The result showed that time after alcohol fermentation is the most suitable time for filtering to remove waste. At that time, concentration of ethanol was the same among the samples. However, concentration of polyphenol, vitamin C, color intensity were higher than in the sample filtered before alcohol fermentation and but stayed the same as sample filtered after acetic fermentation. It showed that during the time enzyme treatment, dry substances in “tao meo” were not totally extracted, during alcohol fermentation, additional extraction may have occurred. However, the extracted sample after acetic acid fermentation was shown with lower concentration of acid than the sample extracted after alcohol fermentation. It may be due to dissolved oxygen process. Sediment in the acetic acid fermentation broth obstructs the dissolving of oxygen and lead to the decrease of transformed fermentation rate. Thus, the better time to remove waste after alcohol fermentation was chosen for vinegar production. Nguyen Thi Viet Anh, Bui Thi Thuy Ha 302 3.2. “Tao meo” extraction processing by pectinase From the results shown in 3.1 paragraph, we chose the treatment method by using both of PectinexUltra SP-L and Pectinex Ultra Clear enzymes for whole experiments. In order to determine suitable parameters for enzyme processing procedure, supplemented ratio of enzyme, hydrolysis temperature and time was estimated. 3.2.1. PectinexUltra SP-L concentration and hydrolysis time for “Tao meo” processing “Tao meo” crushing after dilution was supplemented with Pectinex UltraSP-L at different concentration and the hydrolysis time. Hydrolysis temperature was maintained at 30 o C. When supplement enzyme at different concentration, in different hydrolysis time, galacturonic acid (AUA) amount obtained was different. The result shown in Table 3 indicated that the highest pectin hydrolysis was obtained when the amount of enzyme was 0.15% and the hydrolysis time was 60 min. This result was used in next experiments. Table 3. Effect of concentration enzyme and hydrolysis time on pectin hydrolysis in “tao meo” juice. Enzyme cont. (%) Anhydrogalacturonic acid content (AUA) (%) 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120 min 0.05 0.0110 0.058 0.112 0.163 0.178 0.169 0.156 0.154 0.1 0.0127 0.072 0.153 0.225 0.218 0.219 0.216 0.213 0.15 0.0123 0.087 0.164 0.227 0.226 0.224 0.217 0.212 0.2 0.0125 0.079 0.161 0.214 0.215 0.208 0.197 0.191 0.25 0.0119 0.071 0.158 0.192 0.209 0.205 0.195 0.188 3.2.2. Hydrolysis temperature of PectinexUltra SP-L for “Tao meo” processing “Tao meo” crushing juice was supplemented with Pectinex UltraSP-L at different the hydrolysis temperature. Hydrolysis time was maintained for 60 min with 0.15 % concentration of enzyme. When maintain enzyme activity in different hydrolysis temperature, galacturonic acid (AUA) amount obtained was different. The result shown in Table 4 indicated that the highest pectin hydrolysis result was in 30 o C. This result was used in next experiments. Table 4. Effect of temperature of Pectinex Ultra SP-L on pectin hydrolysis ability in “Tao meo” juice. Hydrolysis temperature ( o C) 25 30 35 40 AUA (%) 0.078 0.236 0.213 0.087 Study on treatment technology of “Tao meo” using pectinase enzyme 303 3.2.3. Concentration of PectinexUltra Clear and hydrolysis time for hydrolysis procedure of “Tao meo” “Tao meo” juice was continuously treated with Ultra Clear enzyme after being treated with Ultra SP-L enzyme. This is pectinase which usually used in clarification of fruit solution because of its higher pectin hydrolysis activity and deeper cleavage of polypectin branches than Ultra SP-L enzyme. Table 5. Effect of PectinexUltra Clear concentration and hydrolysis time on pectin hydrolysis ability in “Tao meo” juice. Enzyme Cont. (%) Clarity level (at OD 620 nm) Color intensity ( at OD 430 nm) 30 min 45 60 75 90 30 min 45 60 75 90 0.05 0.45 0.41 0.39 0.40 0.40 0.12 0.19 0.15 0.15 0.14 0.1 0.21 0.19 0.13 0.14 0.15 0.23 0.28 0.34 0.32 0.33 0.15 0.23 0.21 0.17 0.17 0.19 0.22 0.25 0.30 0.29 0.25 0.2 0.24 0.26 0.24 0.22 0.23 0.24 0.28 0.31 0.32 0.32 Ultra Clear enzyme was added in fruit juice with different concentration and hydrolysis time, hydrolysis temperature was maintained at 55 o C. At the end of experiment, enzyme was unactivated at 85 o C for 5 min. Solution was filtered, settled for 30 min then its color intensity was measured. Samples were stored for 7 days at 4 o C, then the clarity was measured at the wavelength of 620 nm. The result is shown in Table 5. The result showed after using the second enzyme, ability of “tao meo” juice clarification showed the best at the concentration of 0.1 % with hydrolysis time of 60 min. The affect of hydrolysis temperature was studied. The result showed that 55 o C was the best hydrolysis temperature for Pectinase Ultra Clear (data not shown). 3.3. Effect of enzyme treatment on “Tao meo” vinegar quality The result showed the treatment of “tao meo” by both pecticnases increased extraction efficiency as well as fruit juice quality in both functional ingredients and color. However, to fully evaluate the role of enzyme treatment procedure, it is necessary to evaluate the effect of this procedure on process of alcohol and acetic acid fermentation. After hydrolization, whether dissolved pectin concentration affected the clarity of product during storage. Some research indicated that using of pectinase had good influence on extraction and clarification efficiency, but showed some disadvantage on alcohol process such as increase in methanol concentration [7]. The reason is the cleavage of este linkage in methoxy group of polypectin by pectinase that produced methanol [8, 9]. However the produced methanol concentration is low which has no effect on wine quality. Beside these researches on wine products, it is not found any research on the effect of pectinase treatment procedure in vinegar production. In this paper, we paid attention on effect in clarity and quality of “Tao meo” vinegar after fermentation with pectinase treatment. About the transformation of methanol compound, high alcohol compound in wine and vinegar fermentation was not mentioned in this paper. Quality of “tao meo” vinegar produced by different methods is shown in Table 6. Nguyen Thi Viet Anh, Bui Thi Thuy Ha 304 Table 6. Effect of hydrolyse pectin method to “Tao meo” quality. Treatment method Main components of “Tao meo” vinegar Total acid (g/l) Polyphenol (g/l) Vitamin C (g/l) Pectin (%) Potassium (g/l) Total sugar (g/l) Color intensity Without enzyme 30 0.18 1.4 0.125 0.31 4.9 0.18 Ultra SP-L 34 0.24 1.6 0.219 0.39 5.0 0.29 UltraSP- L+UltraClear 36 0.27 1.75 0.242 0.43 5.0 0.35 The data indicated that the treatment procedure of pectinase had no effect on fermentation of “Tao meo” vinegar. On the other hand, concentration of acid and functional compound such as polyphenol, vitamin C, potassium were even higher than control sample, where the sample treated by both enzymes showed best result. The enzyme-treated samples had brown yellow or light brown color which was better than non-treated sample. When comparison our final product sample with two apple vinegars of Mizuka (Japan), it showed the same acid components and vitamin C between them. Polyphenol and potassium concentrations in our final product sample was higher than in Japan vinegar. The reason maybe higher concentrations of polyphenol and potassium in “Tao meo” than in apple used in Japan vinegar product (data not shown). Pectin concentration in product which treated by both enymes was higher than in the sample treated with only one enzyme and significantly higher than the control sample. This is disadvantage for storage procedure if pectin components link together and make sediment in product. The experiment evaluated the change of pectin component of product in 6-month storage. The result is shown in Fig. 1. Firgure 1. Effect of treatment pectinase method to pectin component of vinegar during storage. Pectin concentration in experiment used both enzymes showed better maintenance than two-other experiments, that indicated better cleavage of pectin in solution into monomolecules which stayed in steady dissolved state and rarely revert to make sediment in product. Without treatment of enzyme, pectin concentration in the sample decreased fast, sediment appeared in product just after two- month storage. In one-enzyme-treated sample, although extration efficiency was nearly same as in two-enzyme- treated sample, incompleted hydrolysis leaded to the high existence of pectin which is time-dependent change that make sediment after six-month storage. 4. CONCLUSION The obtained results have shown that the application of pectinase during the manufacturing of “tao meo” vinegar has increased the extractivity of fruits as well as the following quality of Study on treatment technology of “Tao meo” using pectinase enzyme 305 “Tao meo” vinegar such as: amounts of acid, polyphenol, vitamin C (compared to vinegar without enzyme treatment), the appearance of products during storage (color, transparency). Using the two- pectinases as PectinexUltra SP-L and PectinaseUltra Clear during the processing stage, has yielded better results compared to that of using only PectinexUltra SP-L. These results may be applied to the ”tao meo” vinegar technology processing by submerged fermentation method on an industrial scale and aiming at producing function drinks that excel in nutritious, appearance, and food safety qualities. The effect of the enzyme treatment stage on high alcoholic substances and the amount of methanol during the fermentation of “tao meo” vinegar is still currently under investigation and improvement. REFERENCES 1. Trần Đ. T., Nguyễn V. T. - Phát triển cây Sơn tra (táo mèo) ở tỉnh Yên Bái, Tạp chí Khoa học và Công nghệ Đại học Thái Nguyên (92) (4) (2012) 75-81 2. Viện Công nghiệp thực phẩm: Nghiên cứu công nghệ sản xuất đồ uống chức năng từ quả táo mèo bằng phƣơng pháp lên men axetic sử dụng vi khuẩn Acetobacter, Đề tài cấp Bộ Công Thƣơng, 2011, tr. 15-32. 3. Ignacio O., Luis E. R., Domingo C. - Optimum starting-up protocol of a pilot plant scale acetifier for vinegar production, Journal of Food Engineering 52 (2002) 31-37 4. Yoshikatsu M., Mitsuo Y. - Traditional healthful fermented products of Japan, J. Ind Microbiol Biotechnol 35 (2008) 791-798. 5. Esra C., Ric V., Robert. D. H., Dilek Bo., Jules B. - Changes in polyphenol content during production of grape juice concentrate, Food. Chemistry. 139 (2013) 521-526. 6. Arianna R., Dante M. D. F., Simone G., Roberta D., Milena L. - Efect of pre-treaments on the saccharification of pineapple waste as a potential source for vinegar, J. of Cleaner Production 112 (2016) 4477-4484. 7. Hui Z., Edwward E. W., Yong D. H. - Influence of pectinase treatment on spirits from apple mash, juice and pomase, Process Biochemistry 46 (10) (2011) 1909-1913. 8. Indu P., Vasantha R. - Bio-conversion of apple pomace into ethanol and acetic acid, enzymatic hydrolysis and fermentation, Bioresource Technology 130 (2013) 613-620. 9. Manuel P., Birgitte Z., Anne S. M. - Juice clarification by protease and pectinase treatment indicates new roles of pectin and protein in chery juice, Food and Bioproducts Processing 88 (2) (2010) 259-265. TÓM TẮT NGHIÊN CỨU KỸ THUẬT XỬ LÝ TÁO MÈO SỬ DỤNG ENZYME PECTINASE ỨNG DỤNG CHO SẢN XUẤT GIẤM BẰNG PHƢƠNG PHÁP LÊN MEN CHÌM Nguyễn Thị Việt Anh*, Bùi Thị Thúy Hà Viện Công nghiệp Thực phẩm, 301 Nguyễn Trãi, Thanh Xuân, Hà Nội, Việt Nam * Email: vietanhjp27@yahoo.com Nguyen Thi Viet Anh, Bui Thi Thuy Ha 306 Giấm táo mèo có nhiều công dụng tốt cho hệ tiêu hóa, tim mạch, miễn dịch, đƣợc tiêu thụ dƣới dạng đồ uống chức năng. Tuy nhiên, sản phẩm giấm táo mèo chủ yếu đƣợc sản xuất thủ công, theo phƣơng pháp lên men bề mặt, tiềm ẩn nhiều mối nguy. Enzyme pectinase đƣợc sử dụng nhiều trong sản xuất dịch quả quy mô công nghiệp, làm tăng chất lƣợng sản phẩm, tăng hiệu suất trích li, không tạo cặn trong quá trình tàng trữ. Trong bài báo này, đã tập trung nghiên cứu ứng dụng đồng thời hai loại enzyme: PectinexUltra SP-L và PectinaseUltra Clear (Novozyme) cho xử lí quả táo mèo. Kết quả khi sử dụng đồng thời hai loại enzyme đã nâng cao hiệu suất trích li lên 25 %, hàm lƣợng các chất chức năng nhƣ: vitamin C, polyphenol, đƣờng, acid, pectin hòa tan tăng lên đáng kế so với không dùng enzyme. Phƣơng pháp trích ly dịch quả bằng hai loại enzyme đƣợc xác định, đối với enzyme PectinexUltra SP-L: nồng độ là 0,15 %, nhiệt độ thủy phân: 30 oC, thời gian thủy phân: 60 phút; và enzyme PectinaseUltra Clear:nồng độ là 0,1 %, nhiệt độ thủy phân: 55 oC, thời gian thủy phân: 60 phút là thích hợp. Công đoạn xử lí enzyme không ảnh hƣởng tới quá trình lên men giấm, giấm táo mèo sau khi xử lí bằng 2 loại enzyme chứa hàm lƣợng poplyphenol, vitamin C, kali cao hơn so với mẫu không xử lí enzyme, quá trình lọc nhanh hơn. Hàm lƣợng pectin hòa tan trong mẫu giấm táo mèo xử lí enzyme không biến đổi, không tạo cặn trong 6 tháng tàng trữ so với mẫu không xử lí enzyme tạo cặn sau 2 tháng tàng trữ. Từ khóa: giấm táo mèo, pectinase, polyphenol, đồ uống chức năng.