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MindMap Gallery Response surface optimization of extraction process and purification of soy protein isolate

Response surface optimization of extraction process and purification of soy protein isolate

This is a mind map about response surface optimization of the extraction process and purification of soy protein isolate. The main content includes: references, materials and methods, abstract, conclusion, results and analysis, and introduction (the purpose and significance of the topic selection).

Edited at 2024-02-16 20:56:57

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Response surface optimization of extraction process and purification of soy protein isolate

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Outline

Response surface optimization of extraction process and purification of soy protein isolate

Summary

Preface (the purpose and significance of the topic)

Materials and Methods

Materials, reagents and instruments

Material

Soybeans, commercially available

instrument

Type 752 spectrophotometer; Type UV-265 UV-visible recording spectrometer; Type PHS-3C pH meter; SCIENTZ-18N freeze dryer

Reagents

β-Mercaptoethanol, Coomassie Brilliant Blue G-250, acrylamide, SDS

Experimental methods/operations

Sample Preparation

Extraction of soy protein isolate

Alkali extraction and acid precipitation

At room temperature, mix soybean meal with water in a ratio of 1:15 and stir for 1 hour in a pH 8.0 environment to dissolve the protein. The pulp residue was then separated by centrifugation at 3000 r/min for 15 minutes and the precipitate was discarded. Adjust the supernatant to pH 4.5 and let it stand for 30 minutes to precipitate (use alkaline to acidic conditions to precipitate soy protein). Then centrifuge again for 25 minutes to collect the precipitate, wash it three times with deionized water and dissolve it in five times the volume of distilled water. Finally, the pH of the solution was adjusted to 7.5, and the third high-speed centrifugation (30,000 r/min, 25 min) was performed. The precipitate obtained after removing the supernatant was the initially extracted crude soy protein isolate.

Calculation of protein extraction rate (calculated on dry sample)

Protein extraction rate = content of extracted soy protein / protein content in soybeans × 100%

Establishment of standard curve

Sample protein content determination

Prepare a series of standard protein solutions with known concentrations, react with Coomassie Brilliant Blue dye, measure the absorbance and draw a standard curve. Process the protein sample to be tested to an appropriate concentration and incubate it with the dye under stable conditions. Measure the absorbance of each tube at 595nm wavelength, including blank control and sample. Use the obtained absorbance value to calculate the protein concentration in the sample with reference to the standard curve. Ensure constant conditions during operation, paying attention to the effects of light, temperature, and differences in sensitivity of different protein types to the method.

single factor test

Leaching pH 8

Material-to-liquid ratio 1:14g/mL

Extraction temperature 50℃

Extraction time 60min

Conduct response surface optimization experiments based on the above conditions.

Response surface testing

Three levels and four factors

gel filtration

Prepare the agarose gel by wet method and fill it into the chromatography column, and use 1mol/L NaCl PBS buffer (the buffer is prepared from 50mmol/L KH2PO4-Na2HPO4, pH value is 7.6) for equilibrium treatment. The sample needs to be prepared in PBS buffer. After centrifuging to remove the supernatant, ensure that the concentration of the protein solution is less than 10mg/mL, because experiments have repeatedly proven that this concentration can achieve high adsorption rate and short equilibrium time. Next, put the sample on the column, use phosphate buffer for linear gradient elution, set the flow rate to 1.0mL/min, collect one tube every 6mL, and use a UV detector to detect at a wavelength of 280nm. Finally, the collected samples were freeze-dried and analyzed by SDS-PAGE method.

SDS-PAGE detection

According to the method of Kasran et al. [15], SDS-PAGE analysis was performed on the soy protein isolate that had been gel-filtered. The separating gel accounted for 15% and the stacking gel accounted for 5%.

The selected relative molecular mass standard proteins include: phosphorylase (97400 Da), bovine serum albumin (66200 Da), rabbit actin (43000 Da), bovine carbonic anhydrase (31000 Da), trypsin inhibitor (20100 Da) and egg white lysozyme (14400 Da).

results and analysis

Single factor test results

Effect of extraction pH 8 on extraction rate

Effect of solid-liquid ratio 1:14g/mL on extraction rate

Effect of extraction temperature 50℃ on extraction rate

Effect of extraction time of 60 minutes on extraction rate

Response surface optimization experiment

Response surface optimization experimental design and results

It can be seen from the table: the model F value is 5.65, P < 0. 0024, indicating that the model is significant and the fit is good; the lack of fit item P = 0.0737 > 0. 05, indicating that the model has no significant difference, small error, and high fitting degree. In addition, the R2 of the model reaches 0.8682, which can explain 86.62% of the variation in response values, indicating that the predicted results are highly consistent with the actual results. R2adj = 0.7145 indicates that 71.45% of the soybean protein isolate extraction conditions can be used for data analysis. The degree of influence of various factors on the extraction of soybean protein isolate is A > C > D > B, that is, extraction pH > extraction temperature > extraction time > material-liquid ratio. A and C have extremely significant effects (P ＜ 0. 01), D, A2, and C2 have significant effects (P ＜ 0. 05), and other items have no significant effects.

Verification experiment

Through response surface analysis, the optimal extraction process conditions were obtained as extraction pH 9.00, solid-liquid ratio 1:15g/mL, extraction temperature 57.00℃, and extraction time 64.01min. In order to facilitate actual operation, the optimal extraction process conditions were adjusted to extraction pH 9, solid-liquid ratio 1:15g/mL, extraction temperature 57°C, and extraction time 64 minutes. A verification experiment was conducted under these conditions, and the average extraction rate was measured to be 4.75, which is close to the predicted value. The RSD of 0.98% shows that the response surface test results are reliable.

gel filtration chromatogram

SDS-PAGE detection

in conclusion

Single factor experiments and response surface optimization experiments were conducted on the extraction process conditions of soybean protein isolate from soybean meal. The optimal process conditions obtained were: extraction pH 9, solid-liquid ratio 1:15g/mL, extraction temperature 57°C, and extraction time. 64 minutes. Under optimal conditions, the extraction rate of soy protein isolate was 82.58%, and the protein content in the extract was 92.65%. After gel chromatography, the target protein spectrum obtained showed a single elution peak, and the total protein content of the purified SPI reached 98.56%. It was then verified by SDS-PAGE that there were only a few mixed bands in the electrophoresis pattern, indicating that the protein had a high purity and could meet the needs of subsequent scientific research experiments.

references

[1] Han Jing, Zhang Qing'an, Yang Xiaoying, et al. Optimization of ultrasonic-assisted extraction of protein from bean dregs using response surface methodology [J]. Agricultural Products Processing (Part 2), 2015(9):4-8.

[2] Lu Chen, Zou Yuhong, Zhang Shikang, et al. Optimization of process conditions for ultrasonic-assisted extraction of tea residue protein using response surface methodology [J]. Journal of Food and Biotechnology, 2012(3): 319-325.

[3] Lu Chen, Zou Yuhong, Zhang Shikang, et al. Optimization of process conditions for ultrasonic-assisted extraction of tea residue protein using response surface methodology [J]. Journal of Food and Biotechnology, 2012(3): 319-325.

[4] Lang Jiaxue, Liu Chang, Zhao Miao, et al. Research on optimizing the hydrolysis of soybean okara protein by alkaline protease using response surface methodology [J]. Food Research and Development, 2022, 43(3): 129-135.

[5] Guo Xingfeng, Zhang Juanjuan, Ma Yuxiang, et al. Research on optimization of neutral protease extraction of protein from rice residue using response surface method [J]. Journal of Henan University of Technology: Natural Science Edition, 2008, 29(6): 13-17.

[7] An Chuanxiang, Bi Yuanlin, Pei Puhua, et al. Optimization of protein extraction process from walnut meal using response surface methodology [J]. Food Research and Development, 2017, 38(22): 38-43.

[8] Gao Li, Cai Xiaofeng, Li Jing. Extraction, purification and identification of soybean protein isolate [J]. China Food Additives, 2022, 33(11): 136-141. DOI: 10.19804/j.issn1006-2513.2022.11.018.

[9]Kasran M, Cui S W, Goff H D. Emulsifying properties of soy whey protein isolate-fenugreek gum conjugates in oil-in-water emulsion model system［J］. Food Hydrocolloids, 2013, 30(2): 691-697.

Team member: Ao Jian