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1、Application of Protease to Accelerate Sufu Ripening Wang Jianming*1, Wan Shoupeng2, Gu Jinying1 1Key Laboratory of Food Nutrition and Safety, Ministry of Education Tianjin University of Science and Technology Tianjin, China, 300457 wjm-; crab_ 2 Tianjin Limin Condiment Co., Ltd. 226, west 14 Road, T
2、ianjin Airport Industrial Park, Tianjin, China, 300308 Abstract-Sufu is a series of fermented soybean products originated in China, which has a cheese-like texture and special flavor in orient food dishes. However, the disadvantages of high salt content and long ripen time holdback this famous soyb
3、ean food from wide consumption and fast development in modern world. The object of this research was to investigate the effect of enzymatic acceleration sufu ripen period according to the concentration of amino acid nitrogen released and the sensory characteristics produced. The optimized enzymatic
4、acceleration was obtained when 13% soya curd was proteolysis by 2.5% protease at the temperature of 50 for 5 d and then inactivated at 100 for 20min to denature the enzyme activity. Sensory evaluation of the final product showed that the quality of this novel sufu product was closely similar to the
5、traditional one, which indicated the feasibility to accelerate sufu production by enzyme application. Keywords-sufu; ripen; proteinase; acceleration I. INTRODUCTION Sufu (fermented bean curd) or furu in hieroglyphics, it has a soft cheese-like texture with a spreadable creamy consistency and a prono
6、unced flavor. Compared with cheese, sufu has the similar principle and technology in protein coagulation and degradation, similar manufacture procedure and parameters, as well as the similar nutritional unitization and consumed style, thus has the honor of the orient vegetal cheese. Similar to tradi
7、tional ripen cheese process, the period of ripening process is also needed in sufu production to form special flavor and aroma of the final products. Conventionally, the ripen period is slow and costly, about 2 to 6 months or even longer 1. In traditional sufu, the higher salty content plays a key r
8、ole in the anti-corrosion, which, at the same time, stops modern people from large consumptions. For this reason, sufu producers want to reduce salt levels by shortening processing period without change of the flavor to the final product. Amino acids contributing directly to sufu flavor and aroma co
9、mpounds are obtained during sufu ripening 2. Quantification of the free amino acids (FAA) can be quite useful for sufu undergoing accelerated ripening for the purpose of evaluating their effect on flavor and aroma intensity 3; certain FAA are extremely important factors in characteristic flavor deve
10、lopment. Proteolysis is a major event in the ripening of most sufu varieties. Many changes can be perceived during sufu ripening and practically mostly of them can be measured by any of the available methods. Amino acid nitrogen under defined conditions is probably the most widely used as criterion
11、of proteolysis. Hence research on accelerated ripening has focused mainly on the use of protease. The object of this research was to investigate the effect of enzymatic acceleration sufu ripen period according to the concentration of amino acid nitrogen released and the sensory characteristics produ
12、ced and so as to evaluate the feasibility of accelerating sufu production by protease application. II. MATERIALS AND METHODS A. Preparation Soybean, salt, ethanol purchased in the market; P.camemberti stored in Microbiology Lab of Tianjin University of science and technology; composite protease from
13、 Jining Nature Foods Co., Ltd. Sufu Manufacture Traditional Sufu manufacture: Soy milk preparation Pehtze preparation mold fermentation salting ripening traditional sufu Expeditious Sufu manufacture: Soy milk preparation addition of enzymes pehtze preparation mold fermentation expeditious sufu B. Ex
14、perimental Design Single factor trails were designed to investigate the effects comprised substrate (from 10% to 15%, 1% interval), composite protease (from 0% to 3.5%, 0.5% interval), temperature (from 30 to 60, 5 interval), respectively, on amino acid nitrogen. Based on single factor trails, we de
15、signed L9 (34) orthogonal trail to determine the optimum conditions of ripen process after adding enzymes, as shown in Table 1. TABLE 1. FACTORS LEVEL IN L9(34) ORTHOGONAL EXPERIMENTS A B C Substrate (%) Temperature () Protease (%) Level 1 12 40 2.0 Level 2 13 45 1.5 Level 3 14 50 2.5 978-1-4244-471
16、3-8/10/$25.00 2010 IEEE1) Physical and Chemical Analyses Sufu samples were analyzed for amino acid nitrogen by formaldehyde titration, salt by chloride analysis, crude fat by Soxhlet extraction and titrated total acids 4. Samples were selected to measure total acidity, amino acid nitrogen, salinity,
17、 crude fat, pH and other indicators, respectively. 2) Flavor Evaluation Sufu samples were sensory evaluated for appearance, flavor, body and texture by ten panelists. Intensity of each attribute was scored according to an increasing scale from 1 to 10 (Table 2). In order to obtain a final score that
18、 would accurately reflect sufu quality, evaluation was implemented three times by different orders, and the average score was the final result. The maximum overall score was 10. Meanwhile, volatile compounds were analyzed by GCMS detection. TABLE 2. CRITERION OF SENSORY SCORE Characteristic Score Fl
19、avor and smell Strong smell, remain a little aftertaste, ethanol flavor unconspicuous 5 Stronger smell, remain a little aftertaste, ethanol flavor appreciably 4 Flat smell, ethanol flavor appreciably 3 Flatter smell, no aftertaste, ethanol flavor stronger 2 Flatter smell or none, ethanol flavor stro
20、nger, and other mixed flavor 1 Structure and color Uniform structure, surface gloss, uniform color 5 Uniform structure, surface gloss, slight dark of color 4 Uniform structure, worse surface gloss, a little disuniform color 3 Uniform structure, have pellet feeling, disuniform color 2 Ununiform struc
21、ture, worst surface gloss, ununiform color 1 III. RESULTS AND DISCUSSION C. Single-factor Experiments 1) Substrate In sufu production, soy protein plays an important role in flavor and texture formation of the final products. Thus the soybean content can affect the quality of products as well as the
22、 efficiency of the sufu production. By single factors experiments designed, the effect of substrates content on enzyme acceleration was firstly studied as shown in Figure 1, where the amino acid nitrogen concentration was utilized as the indicator to the proteolysis. 0.400.450.500.550.600.650.704.04
23、.44.85.25.66.0Substrate(%)Amino Acid Nitrogen(%) Figure 1. Substrate effect on amino acid nitrogen As shown in Figure 1, when the substrate was about 11%-13%, the amino acid nitrogen increased in a large extent and reached the peak at the content of 13%, where in amino acid nitrogen was about 0.65%
24、averaged by three parallel trials. And no more amino acid nitrogen was detected when more substrate was added. This phenomenon (Figure 1) indicated that high substrate was in favor of enzyme reaction. However, excessive substrate would result in high viscosity of the liquid and exhaustion of the pro
25、tease, which led to the stagnancy of the enzyme-reaction. Accordingly, we choose the substrate content of 13% as the optimized qualification in the following investigations. 2) Protease In cheese production, addition of enzymes to cheese milk 5 or curd were prove to accelerate the chemical reactions
26、 in cheese maturation and reduce the ripen time required for traditional cheese manufacture 6,7. It was also reported that protease could improve the amino acid nitrogen content in the period of sufu post-production 8. Here in our research, the quantity of the protease added into the reaction was st
27、udied so as to improve the efficiency of the proteolysis, as shown in Figure 2. 0.400.450.500.550.600.650.700.00.51.01.52.02.53.0Protease(%)Amino Acid Nitrogen(%) Figure 2. Protease effect on amino acid nitrogen. From Figure 2 we could learn that when the enzyme was more, the amino acid nitrogen pro
28、duced tended to go up sharply till the protease concentration reached 2.0%, and when the protease was over 2.0%, the curve run to flat, in a sense that enzyme reaction reached saturation, which indicated that in the proteolysis period, the enzyme addition was optimized as 2.0% (w/w). 3) Temperature
29、Sufu manufactured process is very similar to that the cheese, especially the process of ripening. In the cheese manufactured process, ripening temperature influences the extent of proteolysis 9, composition of the cheese manufacture 10, texture 11 and quality 12 of cheese. Here we did the similar re
30、search in sufu to study the effect of temperature on enzyme acceleration as shown in Figure 3. 0.400.450.500.550.600.650.7030354045505560Temperature()Amino Acid N itrogen(%) Figure 3. Temperature effect on amino acid nitrogen The curve in Figure 3 showed obvious upward trend in the range of 40 to 50
31、 and when the temperature increased to 50, the amino acid nitrogen concentration reached the maximum of 0.65%. Afterwards, the degree of proteolysis declined rapidly when the enzyme may be inactivated by higher temperature beyond 55. Accordingly, in the process of enzyme acceleration ripen period, t
32、he temperature of 50 was accepted as appropriate to proteolysis. 4) Duration The ripening period was investigated as shown in Figure 4; where the curve of amino acid nitrogen according to ripening time was described on the accepted parameters of ripen process in the foregoing research. 0.300.350.400
33、.450.500.550.600.650.7012345678Time(d)Amino A cid N itrogen(% ) Figure 4. Influence factor of time According to Figure 4, the amino acid nitrogen produced in proteolysis went up with the duration and kept steady after the ripen time passed five days, where the concentration of amino acid nitrogen pr
34、oduced reached 0.59%, which met the national standard of China sufu product (0.50%). When the proteolysis finished, it was necessary to stop the enzyme from working in the acceleration. Thus the 20 minutes of enzyme deactivation was conducted by heat and the influence of temperature of enzyme deacti
35、vation was determined as shown in Table 3. TABLE 3. INFLUENCE OF TEMPERATURE OF ENZYME DEACTIVATION Temperature () Change of sufu 70 Become black，thick and acerbity 80 Normal but a little black 90 Normal 95 100 Heat is a general method to sterilization and enzyme deactivation. In Table 3, it was ind
36、icted the deactivation temperature of 90-100 was appropriate to keep less damage of final products according to sensory evaluation. D. Orthogonal Trail According to the results of single-factor experiments of enzyme acceleration of sufu ripen, the primary parameters including substrate, protease and
37、 temperature were determined as the significant factor to the ripen process and further orthogonal experiments was designed as shown in Table 1, where the sensory evaluation of the final products was accepted as the indicators to the L9 (34) orthogonal experiments, as shown in Table 4 and the analys
38、is of the results was shown in Table 5 and Table 6. TABLE 4. ORTHOGONAL TRAIL OF ACCELERATING RIPEN BY ADDING ENZYME A (%) B() C (%) Blank Sensory score 1 12 40 2.0 1 3.37 2 12 45 1.5 2 4.13 3 12 50 2.5 3 5.50 4 13 40 1.5 3 5.30 5 13 45 2.5 1 7.50 6 13 50 2.0 2 8.37 7 14 40 2.5 2 7.57 8 14 45 2.0 3
39、7.17 9 14 50 1.5 1 7.61 TABLE 5. RANGE ANALYSIS OF SENSORY EVALUATION A (%) B() C (%) Blank k1 13.00 16.24 18.91 18.48 k2 21.17 18.80 17.04 20.07 k3 22.35 21.48 20.57 17.97 R 9.35 5.24 3.53 2.10 K1 4.33 5.41 6.30 6.16 K2 7.06 6.27 5.68 6.69 K3 7.45 7.16 6.86 5.99 R 3.12 1.75 1.18 0.70 Optimization l
40、evel A3 B3 C3 TABLE 6. VARIANCE ANALYSIS OF SENSORY EVALUATION Variance source Q f S F F table Substrate 17.28 2 8.64 21.61 F0.10（2, 2）=9.0 Temperature 4.58 2 2.29 5.72 F0.05（2, 2）=19.0 Protease 2.08 2 1.04 2.60 Total error 0.80 2 0.40 Total 24.74 8 From Table 5 and Table 6, it was indicated the opt
41、imum combination of the selected experiments factors was A3B3C3, which were substrate14%, protease 2.5% and the proteolysis temperature was 50. Among these three factors, the order of importance on the proteolysis was substrate (A)temperature (B)protease (C). From the Table 6, it was realized that t
42、he key influence factor on sensory evaluation was substrate. E. Physical and Chemical Analyses To make comparison physical and chemical properties of the sufu manufactured by traditional and expeditious methods, the main components of the both samples were analyzed, as shown in Table 7. TABLE 7. COM
43、PARISON OF THE MAIN COMPONENTS (W/W (%) Number Total acids Amino acid nitrogen Salinity Crude fat Expeditious Sufu 1 0.90 0.60 3.39 3.93 2 0.99 0.58 3.45 4.31 3 0.80 0.61 3.41 5.01 4 1.03 0.64 3.40 5.13 5 0.76 0.56 3.37 4.90 6 0.74 0.57 3.38 4.53 7 0.71 0.56 3.43 3.72 8 0.80 0.63 3.36 5.12 9 0.78 0.
44、63 3.39 4.91 Average 0.83 0.60 3.40 4.62 Traditional Sufu 1.02 0.69 12.60 4.94 China National Sufu Standard 1.30 0.35 6.50 From the Table 7, it concluded that the main components of expeditious sufu were similar to the traditional one and met the demands of China National Sufu Standard according to
45、the determination of the indicators to total acids and Amino acid nitrogen content in the final products except the lower salinity, which was consistent to the modern nutritional theory, on the basis of good flavor and quality of the final product. IV. CONCLUSIONS The enzymatic accelerated maturatio
46、n of sufu was investigated by single-factor experiments and optimized by orthogonal design. When the process of proteolysis was conducted as: 13% soya curd as substrate, protease 2.5% and the temperature was 50, when inactivated at 100 for 20min to end the proteolysis of 5 days of ripening, the cont
47、ent of amino acid nitrogen reached 0.65%, which meet the demand of China National Sufu Standard. Phy-chemical analysis along with the sensory evaluation of the final product showed that the quality of this novel sufu product was closely similar to the traditional one, which indicated the feasibility
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