Optimization of Processing Parameters of Stabilizers After Enzymes Hydrolysis for Cloudy Ginkgo Juice

Optimization of Processing Parameters of Stabilizers After Enzymes Hydrolysis for Cloudy Ginkgo Juice Haifeng Yu, Junyan Liu and Jingxi Yang 1 Introduction Ginkgo biloba, dating back 300 million years, is a living fossil. Ginkgo seeds are nutritious, contain starch, protein, lipid, pectin, amino acid, vitamins, trace elements and abundant phenolic compounds and have been used as food and herbal medication in china for several thousand years [1]. Ginkgo biloba is wildly grown in China and the yields of ginkgo seeds are rich. However, they are not made full use. In the daily life, fruit juice is regular consumed, especially cloudy juice. The sensory qualities of cloudy juice are important factors for consumer acceptance [2, 3]. On processing juice, enzymatic hydrolysis is used to obtain higher yield and improved stability [4 6]. Ginkgo juice is studied recently, but there are many sediments in it because of its abundant starch. The major sensory problem is the generation of a large of sediments in ginkgo juice storage. In order to produce stable ginkgo juice, enzymes were used to hydrolyze starch, after that the juice was centrifuged to remove sediment [7]. On the processing, ginkgo juice only has little solids. In other way, stabilizers were added into the juice to improved stability, but the ginkgo juice produced could not stay stability for long time. So the experiment is studied to improve the ability of ginkgo cloudy juice. We find that if stabilizers are added into the juice after enzymes hydrolysis, ginkgo juice would be more stable(data unpublished). Therefore, the aim of this study was to investigate the effect of stabilizers (CMC, pectin and SA) dosage after enzymes hydrolysis on ginkgo cloudy juice and optimize the process conditions by RSM. H. Yu (&) J. Liu J. Yang Shandong Provincial Key Laboratory of Microbial Engineeringy, Qilu University of Technology, No. 3501 Daxue Road, Changqing District 250353 Jinan Shandong Province, People s Republic of China e-mail: yhfdzz@126.com Springer Nature Singapore Pte Ltd. 2018 H. Liu et al. (eds.), Advances in Applied Biotechnology, Lecture Notes in Electrical Engineering 444, https://doi.org/10.1007/978-981-10-4801-2_17 165

166 H. Yu et al. 2 Materials and Methods Ginkgo seeds were purchased from Tancheng city Shandong province. From the pre-experiment we know that the water content is 55.88% (w/w), the protein content is 5.85% (w/w) and the starch content is 28.30% (w/w). 2.1 Enzyme and Stabilizer Source Medium temperature a-amylase and neutral protease were purchased from Ruiyang Biotechnology Company, Jiangsu, China. CMC, pectin and SA were purchased from Quankang Food Ingredients Company, Jinan, China. 2.2 Preparation of Ginkgo Juice Based on pre-experiment: a ratio of 1:20(kernel:water,w/v) was used in the comminuting process; the dosage of enzymes were determined that a-amylase was 0.015 g/100 ml ginkgo juice, neutral protease was 0.02 g/100 ml ginkgo juice, enzyme temperature was 80 ± 1 C and enzyme treatment time was 90 min; the range of the variables for stabilizer conditions was selected, there were CMC dosage, X 1 (0.01 0.13 g/100 ml ginkgo juice), pectin dosage, X 2 (0.02 0.18 g/100 ml ginkgo juice) and SA dosage, X 3 (0.01 0.11 g/100 ml ginkgo juice). The processing of ginkgo juice is showed in Fig. 1. Fig. 1 Flow chart of ginkgo cloudy juice production

Optimization of Processing Parameters of Stabilizers 167 2.3 Centrifugal Sedimentation Rate Ginkgo juice was shaken and certain quality juice was removed to centrifuge tube, the juice was centrifuged at 3000 rpm for 30 min to measure the quality of sediments. The ratio of sediments quality and juice quality was considered a measure of centrifugal sedimentation rate. 2.4 Experimental Design and Statistical Analysis RSM was used in designing this experiment. A Design-Expert V8.0 was used to generate the experimental designs, statistical analysis and regression model. The independent variables were the dosage of CMC (x 1 ).the dosage of pectin (x 2 ), the dosage of SA (x 3 ). Each independent variable had coded levels of 1, 0 and 1. The experimental designs of the coded (x) and actual (X) levels of variables are shown in Table 1. The response (y) is centrifugal sedimentation rate (%). The response function (y) was related to the coded variables (x i, i =1,2,3)bya second-degree polynomial (1) using the method of least squares. Table 1 The RSM experimental design (in coded level of three variables) employed for processing ginkgo cloudy juice with CMC, pectin, SA Experim-ent number CMC (g/100 ml ginkgo juice) Pectin (g/100 ml ginkgo juice) X 1 (x 1 ) X 2 (x 2 ) X 3 (x 3 ) 1 0.13(1) 0.1(0) 0.11(1) 2 0.07(0) 0.18(1) 0.01( 1) 3 0.13(1) 0.18(1) 0.06(0) 4 0.01( 1) 0.1(0) 0.01( 1) 5 0.13(1) 0.1(0) 0.01( 1) 6 0.01( 1) 0.18(1) 0.06(0) 7 0.07(0) 0.1(0) 0.06(0) 8 0.07(0) 0.1(0) 0.06(0) 9 0.07(0) 0.02( 1) 0.11(1) 10 0.13(1) 0.02( 1) 0.06(0) 11 0.01( 1) 0.02( 1) 0.06(0) 12 0.07(0) 0.1(0) 0.06(0) 13 0.07(0) 0.1(0) 0.06(0) 14 0.01( 1) 0.1(0) 0.11(1) 15 0.07(0) 0.18(1) 0.11(1) 16 0.07(0) 0.02( 1) 0.01( 1) 17 0.07(0) 0.1(0) 0.06(0) SA (g/100 ml ginkgo juice)

168 H. Yu et al. y ¼ b 0 þ b 1 x 1 þ b 2 x 2 þ b 3 x 3 þ b 11 x 2 1 þ b 22x 2 2 þ b 33x 2 3 þ b 12x 1 x 2 þ b 13 x 1 x 3 þ b 23 x 2 x 3 ð1þ Analysis of variance (ANOVA) was performed. The significances of all terms were judged statistically by computing the F-value at a probability (p) of 0.001, 0.01 or 0.05.The regression coefficients were used to make statistical calculations to generate contour maps from the regression models. 3 Results and Discussion 3.1 Statistical Analysis The experimental results on the effect of the dependent variables namely CMC dosage, pectin dosage and SA dosage on the response functions are shown in Table 2. The corresponding R 2 and coefficients of the variables in the models are shown in Table 3. The closer the value R 2 is to unity, the better the empirical model fits the actual data. The value R 2 for centrifugal sedimentation rate (after enzymes hydrolysis) and for centrifugal sedimentation rate (no enzymes hydrolysis) were 0.9472 and 0.9574, indicating that the regression models explained the reaction well. The probability (p) values of all regression models were less than 0.05. Table 2 Effect of stabilizers on two dependent variables Experiment number Centrifugal sedimentation rate (%) (after enzymes hydrolysis) Y 1 Y 2 1 9.94 35.03 2 9.85 37.23 3 10.33 36.6 4 9.31 35.9 5 12.42 40.02 6 11.22 40.19 7 5.32 22.44 8 5.33 23.48 9 11.32 40.59 10 10.23 36.19 11 9.96 35.11 12 5.36 23.6 13 7.25 28.19 14 12.17 44.01 15 11.12 39.78 16 8.11 31.06 17 5.58 25.68 Centrifugal sedimentation rate (%) (no enzymes hydrolysis)

Optimization of Processing Parameters of Stabilizers 169 3.2 Centrifugal Sedimentation Rate (After Enzymes Hydrolysis) From Table 3, it is observed that the quadratic terms of CMC dosage (p 0.001), pectin dosage (p 0.01) and SA dosage (p 0.001) had a significant effect on centrifugal sedimentation rate. CMC dosage and SA dosage had a negative interaction effect (p 0.05). Figure 2a describes that the dependence of centrifugal sedimentation rate with CMC dosage and pectin dosage at fixed SA dosage. From Fig. 2a, it is clear that at constant pectin dosage and SA dosage, centrifugal sedimentation rate decreased with CMC dosage at the beginning then increased gradually. Likewise, with the increase of pectin dosage, the centrifugal sedimentation rate decreased gradually first, then increased. Figure 2b presents the variation of centrifugal sedimentation rate with CMC dosage and SA dosage at constant pectin dosage. It is evident that at a fixed CMC dosage and pectin dosage, the centrifugal sedimentation rate of ginkgo juice decreased with SA dosage at the beginning and then increased. Table 3 Regression coefficients, R 2 and p value for the response function Coefficient of the regression equation Centrifugal sedimentation rate (%) After enzymes hydrolysis Centrifugal sedimentation rate (%) No enzymes hydrolysis b0 (intercept) 11.76123 40.98628 b1 74.18611 196.18819 b2 43.44896 108.11927 b3 61.143 182.86633* b11 767.63889*** 1933.26389*** b22 297.42187** 841.36719** b33 971.4*** 2840.9*** b12 60.41667 243.22917 b13 445* 1091.66667* b23 121.25 436.25 R2 0.9472 0.9574 p 0.0011 0.0005 b was the coefficient of polynomial b0 (constant); b1, b2 and b3 (linear effects); b11, b22 and b33 (quadratic effects); and b12, b13 and b23 (interaction effects) 1 CMC dosage, 2 pectin dosage, 3 SA dosage *Significant at p 0.05. **Significant at p 0.01. ***Significant at p 0.001

170 H. Yu et al. Fig. 2 Response surface diagram for centrifugal sedimentation rate (after enzymes hydrolysis) of ginkgo juice as a function of a CMC dosage and pectin dosage (the SA dosage was kept constant at the central point which was 0.06 g/100 ml ginkgo juice) and b CMC dosage and SA dosage (the pectin dosage was kept constant at the central point which was 0.10 g/100 ml ginkgo juice). X 1 was CMC dosage (g/100 ml ginkgo juice). X 2 was pectin dosage (g/100 ml ginkgo juice). X 3 was SA dosage (g/100 ml ginkgo juice) 3.3 Optimization (After Enzymes Hydrolysis) The optimum processing conditions to centrifugal sedimentation rate were investigated. Figure 3 shows the superimposed contour plot for optimization of centrifugal sedimentation rate (after enzymes hydrolysis) keeping the SA dosage constant at the central point and keeping pectin dosage constant at the central point. The zone of optimization, as shown in the superimposed contour plot, depicts CMC dosage to be in the range of 0.05 0.09 g/100 ml ginkgo juice, pectin dosage in the range of 0.06 0.12 g/100 ml ginkgo juice and SA dosage between 0.03 and 0.08 g/100 ml ginkgo juice. 3.4 Centrifugal Sedimentation Rate (No Enzymes Hydrolysis) It is clear from Table 3 that the linear term of SA dosage (p 0.05) had a negative effect on centrifugal sedimentation rate. The quadratic terms of CMC dosage (p 0.001), pectin dosage (p 0.01) and SA dosage (p 0.001) had a significant effect on centrifugal sedimentation rate. CMC dosage and SA dosage had a negative interaction effect (p 0.05). Figure 4a describes that the dependence of centrifugal sedimentation rate with CMC dosage and pectin dosage at determined SA dosage. From Fig. 4a, it is shown that at constant pectin dosage and SA dosage, centrifugal sedimentation rate

Optimization of Processing Parameters of Stabilizers 171 Fig. 3 Superimposed contour plots for optimization of centrifugal sedimentation rate (after enzymes hydrolysis) when SA dosage was kept constant at central point (0.06 g/100 ml ginkgo juice) (a) and CMC dosage and SA dosage (the pectin dosage was kept constant at the central point which was 0.10 g/100 ml ginkgo juice) (b). X 1 was CMC dosage (g/100 ml ginkgo juice). X 2 was pectin dosage (g/100 ml ginkgo juice). X 3 was SA dosage (g/100 ml ginkgo juice) Fig. 4 Response surface diagram for centrifugal sedimentation rate (no enzymes hydrolysis) of ginkgo juice as a function of a CMC dosage and pectin dosage (the SA dosage was kept constant at the central point which was 0.06 g/100 ml ginkgo juice) and b CMC dosage and SA dosage (the pectin dosage was kept constant at the central point which was 0.10 g/100 ml ginkgo juice). X 1 was CMC dosage (g/100 ml ginkgo juice). X 2 was pectin dosage (g/100 ml ginkgo juice). X 3 was SA dosage (g/100 ml ginkgo juice) decreased with CMC dosage at the beginning then increased gradually. With the increase of pectin dosage, the centrifugal sedimentation rate decreased gradually first, then increased. Figure 4b presents the variation of centrifugal sedimentation rate with CMC dosage and SA dosage at constant pectin dosage. It is evident that at a certain CMC dosage and pectin dosage, the centrifugal sedimentation rate of ginkgo juice decreased with SA dosage at the beginning and then increased.

172 H. Yu et al. 3.5 Optimization (No Enzymes Hydrolysis) Figure 5 shows the superimposed contour plot of centrifugal sedimentation rate (no enzymes hydrolysis) keeping the SA dosage constant at the central point and keeping pectin dosage constant at the central point. The zone of optimization, as shown in the superimposed contour plot, depicts CMC dosage to be in the range of 0.05 0.09 g/100 ml ginkgo juice, pectin dosage in the range of 0.06 0.12 g/100 ml ginkgo juice and SA dosage between 0.03 and 0.07 g/100 ml ginkgo juice. Keeping the CMC dosage constant as firmed from Fig. 5, the best combination of response functions, SA dosage was determined. The process variables for best combination of response functions were CMC dosage 0.07 g/100 ml ginkgo juice, pectin dosage 0.09 g/100 ml ginkgo juice and SA dosage 0.05 g/100 ml ginkgo juice. Fig. 5 Superimposed contour plots for optimization of centrifugal sedimentation rate (no enzymes hydrolysis) when SA dosage was kept constant at central point (0.06 g/100 ml ginkgo juice) (a) and CMC dosage and SA dosage (the pectin dosage was kept constant at the central point which was 0.10 g/100 ml ginkgo juice) (b). X 1 was CMC dosage (g/100 ml ginkgo juice). X 2 was pectin dosage (g/100 ml ginkgo juice). X 3 was SA dosage (g/100 ml ginkgo juice)

Optimization of Processing Parameters of Stabilizers 173 4 Conclusion Using RSM, the optimum condition was obtained. These were CMC dosage 0.07 g/100 ml ginkgo juice, pectin dosage 0.09 g/100 ml ginkgo juice and SA dosage 0.05 g/100 ml ginkgo juice (the optimum condition was the same between after enzymes hydrolysis and no enzymes hydrolysis processing for ginkgo juice). Enzymes complete the hydrolysis of starch into low-molecular substances relative to starch. After that, put stabilizers into ginkgo juice, these low-molecular substances in juice are more stable. By this experiment, it is known that the lowest centrifugal sedimentation rate is 5.70% for after enzymes hydrolysis and 24.44% for no enzymes hydrolysis processing. From these two figures, it is clear that if ginkgo juice is enzymes hydrolyzed first and then put into stabilizers, we can get much more stable ginkgo juice. For this, you can store ginkgo juice for longer time with little sedimentation. Acknowledgements This work was supported by the Excellent Middle Aged and Young Scientist Award Foundation of Shandong Province (No. BS2011SW029) and a Project of Shandong Province Higher Educational Science and Technology Program (No. J13LE01) and Science and technology development project of Shandong Province (No. 2014GSF121039). References 1. Fu XH, Li F, Xu CQ, Wei X (1997) Studies on growth of fruits and components of endosperm in seeds of Ginkgo biloba cv. Jiangsu DafoShou. GuangXi Plant 17(3):263 269 2. Cameron R (1997) Citrus tissue extracts affect juice cloudy stability. J Food Sci 62:242 245 3. Rubico SM, Resurreccion AVA, Frank JF, Beuchat L (1987) Suspension stability, texture, and color of high temperature treated peanut beverage. J Food Sci 52(6):1676 1679 4. Abastasakis M, Lindamood JB, Chism GW, Hansen PMT (1987) Enzymatic hydrolysis of carrot for extraction of a cloud-stable juice. Food Hydrocolloids 1:247 261 5. Pilnik W, Voragen GJ (1993) Pectic enzyme in food and vegetable juice manufacture. In: Enzymes in food processing. Academic Press, New York, pp 367 371 6. Reiter M, Stuparic M, Neidhart S, Carle R (2003) The role of process technology in carrot juice cloudy stability. Lebensmittle-Wissenschaft Und Technologie 36:165 172 7. Zhang H, Wang Z, Xu S-Y (2007) Optimization of processing parameters for cloudy ginkgo (Ginkgo biloba Linn.) juice. J Food Eng 80:1226 1232

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