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Study on lipids transfer in aqueous enzyme hydrolysis soybean protein and oil extraction process
Industrial Crops & Products 137 (2019) 203–207
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Study on lipids transfer in aqueous enzyme hydrolysis soybean protein and oil extraction process
T
Junqing Qian , Jun Tong, Yan Chen, Shen Yao, Hui Guo, Lanhua Yang ⁎
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, China
ARTICLE INFO
ABSTRACT
Keywords: Enzyme-assisted aqueous extraction Soybean oil Oil transfer Proteolysis Neutral protease Petroleum ether
Enzyme-assisted aqueous Extraction of soybean oil is a promising green and safe alternative to traditional process. However, it is quite difficult to realize industrial application as the process needs large dosage of special enzyme and high-speed separation. To overcome these problems, the quantitative transfer of oil release during the hydrolysis of soybean protein by protease was studied. The results showed that the 1398 neutral protease with the total amount of only 720 IU/g soybean could be used to hydrolyze the soy protein, and the oil yield reached up to 90% after separation by the centrifugal force of 1960 × g. The enzymatic hydrolysis of soybean protein was beneficial to release and separate oil from soybean, and the separated protein could enrich almost 99.0% of oil from hydrolyzed emulsion. At the moment, the emulsification could be eliminated during the process of aqueous extraction with petroleum ether.
1. Introduction Soybean is one of the main edible oil seeds in the world. It is not only used for oil products, but also for the manufacture of high-quality vegetable protein (Loman and Ju, 2017; Ebert et al., 2017). Traditionally, soybean was usually treated at a high temperature to achieve higher oil yield because of low oil content. However, the process of high temperature treatment leads to the serious denaturation of soybean protein, and also affects the quality of oil (Galloway, 1976; Lawhon et al., 1981; Farhadian et al., 2011; Bojanowska and Czerwiński, 2010; Orecchio et al., 2009). For the improvement of the quality of soybean oil and the utilization of soybean protein, the process of enzyme-assisted aqueous extraction has been widely studied. Rosenthala et al. (2001) selected the optimum conditions of aqueous enzymatic extraction of soybean oil by Response Surface Methodology. At the moment, they tested the individual effect of two different enzymes—protease and cellulase—on oil yield with the best process parameters. It was found that protease could significantly contribute to a higher oil yield, 58.7%. Rovaris et al. (2012) explored the relationship between four enzyme systems and soybean oil yield, while the oil yield was only 23%. The amount of enzyme (2.5 wt.%) was increased by Moura et al. (2010) for de-emulsifying of protein emulsion from soybean, while the result was unexpected. The oil yield was improved by adding n-hexane finally. It was reported that pretreatment may improve oil recovery. Jung and Mahfuz (2009) squeezed soybean emulsion at 100 °C and 500 MPa, then
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0.5 wt.% of protease Protex 7 L was added for aqueous extraction, which promoted a significant increase in oil recovery (90%). After evaluating different proteases’contribution to free oil, Protex 6L was selected by Tabtabaei and Diosady (2013) at a concentration of 2.5 wt. % for hydrolysis. Results showed that 91% of oil from the emulsions can be recovered effectively with gradient centrifugation (9000 × g). The above studies on pre-treatment methods such as high temperature and pressure, and the selection of optimal protease showed that almost 90% of soybean oil could enter into the emulsion. The above studies on pretreatment methods such as high temperature and pressure, and the selection of optimal protease showed that almost 90% of soybean oil could enter into the emulsion. Meanwhile, it is crucial to demulsify and separate oil-water two phases for obtaining edible grade soybean oil. Several destabilization methods have been used to release oil from the emulsion formed during enzyme-assisted aqueous extraction of soybean. Protex 50FP and Protex 6L were used for soybean hydrolysis, respectively by Jung et al. (2009) and Wu et al. (2009). Under the optimal condition, nearly 90% of oil entered into the emulsion. Then the emulsion was demulsified by adding salt and organic solvent. Finally, 88% of free oil recovery was obtained. The centrifugal force (10,000 × g) was increased by Towa et al. (2011) after the emulsion treated at 96℃ for the oil yield, and the final yield reached to 90%. Bell et al. (2013) found that soybean oil extracted by enzyme-assisted aqueous extraction has some advantages such as low peroxide value and acid value. A process combing ohmic heating and enzyme assisted
Corresponding author. E-mail address: [email protected] (J. Qian).
https://doi.org/10.1016/j.indcrop.2019.04.063 Received 19 August 2018; Received in revised form 20 April 2019; Accepted 28 April 2019 Available online 20 May 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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aqueous extraction process was investigated by Pare et al. (2014) to improve soy oil recovery, however, the maximum oil recovery (73%) was still lower than other reports. To maximize the free oil yield of soybean oil, ultrasound-assisted enzymatic extraction was developed by Li et al. (2014). A significant efficiency of demulsification was obtained, and the oil recovery reached up to 92.6%. It is the highest oil yield in enzyme assisted aqueous extraction of soybean oil so far. Mechanism of enzyme-assisted aqueous extraction of soybean oil intrigued Han et al. (2015). Although oil recovery was only 69%, they stilled used microscope to intuitively observe the processes of the cells destruction, oil transfer and demulsification during enzymatic hydrolysis of soybean emulsion. Selection of enzymes and demulsification during enzyme-assisted aqueous extraction of soybean oil were studied widly. However, as a vegetable oil extraction method, quantitative studies on oil transfer in enzymatic hydrolysis of soybean emulsion are rarely reported. Although microscopic observation can directly demonstrate the enzymatic hydrolysis process and tissue fragmentation of soybean, it is impossible to obtain the oil transfer quantitatively, which is the basis of extraction process. Some methods such as the selection of outstanding performance enzyme with a dosage up to 2.5 wt.% (Tabtabaei and Diosady, 2013), the increase of centrifugal force (9000 × g) for emulsion separation (Tabtabaei and Diosady, 2013), and the use of ethanol (73%) with ultrasonic treatment for demulsification (Li et al., 2014) helped release oil during the process of enzyme-assisted aqueous extraction. The most highest oil yield could reach up to 92.6%, nevertheless it is not only difficult to industrialize on a large-scale, but unbearable in terms of economic cost for preparation of edible soybean oil. It is necessary to explore a better method of demulsification, which employs an enzyme with suitable performance, lower price and dosage, low speed centrifugation for oil-rich emulsion separation. At the moment, the largescale industrialization and relatively high free oil recovery should be achieved. The quantitative transfer of released oil in the process of protease hydrolysis of soybean protein was studied to overcome above problems, and a process using lower dosage of cheap enzyme for soybean oil extraction was established in this paper. Results showed that the coexistence of petroleum ether and water was beneficial to oil transfer, and had a good effect on demulsification. Therefore, the hydrolysis of soybean protein using 1398 neutral protease with 240 IU/g soybean was explored for the first time to promote the oil diffusion into aqueous phase. The soybean protein with a centrifugal force of 1960 × g was separated by adjusting the pH value of the emulsion,which was enriched 44% oil. Then, oil-enriched soybean emulsion was hydrolyzed by 1398 neutral protease (480 IU/g) once again. Almost 99% of oil could enter into the organic phase after adding petroleum ether. Two phases water and organic were separated with a centrifugal force of 1960 × g, and the emulsification ratio was less than 2.0%. Petroleum ether was recovered by low-vacuum evaporator, and 89.6% of the total oil of soybean was obtained. The acid value of the oil was 2.60 mg KOH/g oil, and the impurity was 0.22%.
Preparative Ultracentrifuge and Hitachi CR20B2 High Speed Refrigerated Centrifuge were used. 2.2. Preparation of soybean aqueous extraction Soybeans (100 g) were crushed to pass through a 20-mesh standard sieve, then soaked in distilled water (400 mL, pH 9.0), and extracted at 50 ℃ for 1.5 h. Next, they were grinded after adding 2 times volume of water, and filtered with 100-mesh sieve. The filter residue was washed by 2 times volume of water. The filtrate was collected for further study. Under this condition, 95.5% (w/w) oil and 95.0% (w/w) protein of soybean entered into aqueous extract emulsion (Qian et al., 2010). 2.3. Research method of lipids transfer in soybean aqueous extract emulsion after enzymatic hydrolysis Soybean aqueous (200 mL) extract emulsion was hydrolyzed by 1398 neural protease at pH 7.0 and 45 ℃ water bath, with stirring rate 300 rpm. Followed by centrifuging at 18,000 × g for 15 min, the system was split two layers (solid phase and aqueous phase) from top to bottom. Followed by centrifuging at 18,000 × g for 15 min, the system was split two layers (solid phase and aqueous phase) from top to bottom. The oil released from soybean protein has a lower density than water, forming upper solid phase. Then the dry basis oil content and total oil of upper solid were determined. Comparing oil release under different conditions, including enzyme dosage and enzymatic time, the effect of enzymatic hydrolysis on the oil release from soybean protein was studied. After enzymatic hydrolysis, pH of the system was adjusted to the isoelectric point of soybean protein. Then it was centrifuged at 1400 × g (15 min) for the soybean protein separation. The dry basis oil content and total oil content of the separated protein were determined to investigate the absorption of separated protein to oil, and evaluate the oil transfer from protein during enzymatic hydrolysis. 2.4. Research method of lipids transfer of soybean emulsion in the presence of petroleum ether Soybean emulsion (200 mL) was hydrolyzed by 1398 neural protease at pH 7.0 and 45 ℃ water bath, with stirring rate 300 rpm. The pH of 200 mL above emulsion was adjusted to 4.5, then 120 mL petroleum ether was added into the solution to extract for 15 min at 30 ℃. The solution was centrifuged at 4420 × g (15 min) for the separation of organic phase from the system. The oil was obtained by evaporation petroleum ether. And the transfer of oil into petroleum ether was investigated. The pH of emulsion separated from petroleum ether was adjusted to 7.0, then the emulsion was centrifuged at 18,000 × g for 15 min. The dry basis oil content and total oil of the upper solid were determined. From this, the release of soybean oil could be compared with the emulsion without petroleum ether. 2.5. Statistical analysis
2. Materials and methods
All analysis was done in triplicate. Data were expressed as the mean ± standard deviation of three replicates. STATISTICA 17 (StatSoft, Tulsa, OK, USA) was applied to conduct the ANOVA test and compare the means of the oil yield at 95% confidence interval.
2.1. Materials Soybeans were purchased from local market (Zhejiang, China). The initial moisture content of soybeans was 12.4% mass (wet basis). The oil content of soybeans was 18.6% mass (dry basis) determined by Soxhlet n-hexane extraction (American Society for Testing and Materials, 2009). The protein content of soybean was 40.5% mass (dry basis). The 1398 Neutral Protease (edible grade) was provided by Wuxi Enzymes Preparation Factory (Jiangsu, China) and the enzyme activity was 120,000 IU/g. All other chemicals and reagents used in this study were analytical grade and commercially available. Beckman LB-55M
3. Results and discussion 3.1. Lipids transfer in soybean aqueous extract emulsion after enzymatic hydrolysis It has been reported that enzymatic hydrolysis is helpful for oil release (Kapchie et al., 2008; Yusoff et al., 2015). According the above 204
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Table 1 Comparison of emulsion release oil with different enzyme dosage. Enzyme dosage (IU/g soybean)
0
240
480
720
960
1200
Released oil
0.65 ± 0.0 56.7 ± 0.1
1.01 ± 0.1 94.4 ± 0.2
1.12 ± 0.0 92.6 ± 0.1
1.09 ± 0.4 92.4 ± 0.5
1.14 ± 0.2 91.9 ± 0.4
1.07 ± 0.2 92.2 ± 1.3
Total oil (g) Oil content of dry basis (%)
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Time of enzymatic hydrolysis is 1.5 h. Values are presented as mean value ± SD (n = 3).
centrifuged at different speed for 15 min, for the study of the effect of centrifuge speed on lipid transfer, results are shown in Table 4. Both results (Tables 3 and 4) suggest that the oil transfer was enhanced after enzymatic hydrolysis. A large number of released oil was adsorbed by proteins which were separated with low-speed centrifugation near isoelectric point, so oil content is greatly improved. The total mass of separated protein and the total content of oil reached to 38% and 94.5% (about 99.0% oil of emulsion) of original soybean oil content, respectively. So it is meaningful for enzyme-assisted aqueous extraction to isolate oil-enriched protein, and this type of phenomenon of lipids transfer in emulsion has never been reported.
paper, emulsion was centrifuged at 18,000 × g after enzymatic hydrolysis, released oil that was less dense to water floated to the top, and the oil binding with protein still remained in the emulsion. Therefore, the effect of enzymatic hydrolysis on oil release under different conditions can be evaluated, through determining the oil content of upper solid and total oil. In order to reduce the protein adsorption to oil, the pH of soybean emulsion was adjusted to 6.0 and 7.0, respectively, according to the isoelectric point (IP) of soybean protein. Then the emulsion was centrifuged for 15 min, and the oil content of upper solid were 84.0% and 93.9%, respectively. Results showed that soybean protein entrapped less oil at pH 7.0, leading to a more accurate evaluation. Finally, the condition of pH (7.0) was selected for further study. Oil release under different enzyme dosages (Table 1) and enzymatic hydrolysis time (Table 2) was determined (each experiment consumed 200 mL emulsion (which oil content is 4.44 g)). From Table 1, the amount of released oil after enzymatic hydrolysis was obviously higher than the solution without enzymatic hydrolysis. The amount of enzyme was up to 480 IU/g soybean, oil content was to be stable, which showed that enzymatic hydrolysis was beneficial to oil release. From both Tables 1 and 2, although enzymatic hydrolysis was helpful for oil release, a small amount of oil was only obtained, and the highest only reached at 26.1% (w/w). Compared with the traditional oil extractions, oil yield appeared to be much lower. In this study, highspeed centrifugation (18,000 × g) was used to separate oil. But with the enzyme dosage increasing, the amount of oil did not increase, and it seems that extending enzymatic hydrolysis time was meaningless. Above all, soybean oil still contained protein (about 7%) after highspeed centrifugation. Results revealed the reason for lower oil yield of enzyme-assisted aqueous extraction which was reported in other literature. Therefore, it was rather difficult to extract oil through enzymeassisted aqueous extraction from vegetable protein. The above experiments confirmed that enzymatic hydrolysis was helpful for oil release, while it was extremely difficult to separate the oil from protein completely. That is, the transfer of lipids in emulsion was enhanced, but the released oil was adsorbed by protein. It doesn’t seem there is a strong connection between oil transfer and oil release. Next, the solids (proteins) were separated from soybean emulsion after enzymatic hydrolysis with low-speed centrifugation, for the study of lipids transfer. According to Table 1, 200 mL emulsion was hydrolyzed in 45 ℃ for 1.5 h at following conditions: enzyme dosage 240 IU/g soybean; pH 7.0; stirring rate 300 rpm. Then, the pH of the solution was adjusted close to isoelectric point before centrifuging (1400 × g). Results of oil content of separated protein are shown in Table 3. From Table 3, we adjusted the pH of solution to 4.5, then it was
3.2. Lipids transfer in soybean emulsion in the coexistence of petroleum ether Enhancing the transfer of lipids in aqueous phase contributes to efficient oil extraction from soybean protein. In the following research, the common method of vegetable oil extraction was introduced, and the oil transfer during the petroleum ether extraction was studied. The oil transfer in the organic phase under different extraction time was investigated, and the oil release of remained protein was evaluated through calculating oil content of upper solid and total oil after centrifuging at 18,000 × g (each experiment consumed 200 mL emulsion). Results are shown in Table 5. From Table 5, we found that 73% oil of emulsion that was extracted by petroleum ether entered into the organic phase. And the number of oil release from remained protein was greater than that was shown in Tables 1 and 2 . The sum of the oil released from enzymatiac emulsion and remained protein nearly reached 100% of the total oil, which indicated that petroleum ether extraction in enzyme-assisted aqueous extraction can obviously promote the oil transfer in internal hydrolyzed protein. The structure of protein was destroyed through enzymatic hydrolysis, leading to a easier lipids transfer. Meanwhile, organic extraction makes it easie to transmit oil from internal protein to the surface. And the extension of extraction time also helped oil transfer outward. After 15 min at equilibrium, the whole oil could be released under high-speed centrifugation. It was a great breakthrough for enzyme-assisted aqueous extraction reported in literature. Better oil transfer has been achieved, while the problem of emulsification needs to be solved. Next, the effect of different centrifugal forces on emulsification after extracting for 15 min was studied and the results were shown in Table 6. Emulsification rate could be controlled less than 2.0%, under the condition of centrifuging at 1960 × g for 15 min. Compared with the methods reported in former literature of demulsificasion, it was easier to be realized.
Table 2 Comparison of emulsion release oil different time of enzymatic hydrolysis. Time of enzymatic hydrolysis (h)
0
0.5
1.0
1.5
2.0
Released oil
0.65 ± 0.0 56.7 ± 0.1
0.75 ± 0.0 93.8 ± 0.1
1.03 ± 0.2 91.2 ± 0.3
1.16 ± 0.1 92.8 ± 0.2
1.14 ± 0.2 92.7 ± 0.4
Total oil (g) Oil content of dry basis (%)
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Enzyme dosage is 480 IU/g soybean. Values are presented as mean value ± SD (n = 3). 205
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Table 3 Oil adsorption by separated protein after hydrolyzed at different pH. pH
3.5
4.0
4.5
5.0
5.5
Dry weight of protein (g) Oil content (%) Percentage of total oil (%)
8.67 ± 0.2 41.9 ± 0.1 81.8 ± 0.2
9.26 ± 0.3 43.7 ± 0.2 91.1 ± 0.0
9.49 ± 0.1 43.8 ± 0.3 93.6 ± 0.1
8.95 ± 0.1 43.2 ± 0.2 87.1 ± 0.1
8.63 ± 0.2 41.5 ± 0.4 80.7 ± 0.3
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Values are presented as mean value ± SD (n = 3). Table 4 Oil adsorption by protein separated at different centrifugal force. Centrifugal force (g)
1100
1400
1960
3070
4420
Dry weight of protein (g) Oil content (%) Percentage of total oil (%)
8.56 ± 0.3 42.3 ± 0.2 81.5 ± 0.0
9.47 ± 0.2 43.9 ± 0.1 93.6 ± 0.2
9.51 ± 0.2 44.1 ± 0.1 94.5 ± 0.1
9.52 ± 0.1 43.2 ± 0.3 92.6 ± 0.2
9.51 ± 0.1 40.6 ± 0.3 87.0 ± 0.0
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Values are presented as mean value ± SD (n = 3). Table 5 Oil transfer of extraction phase and oil release of remained protein with different time of extraction. Extraction phase Oil release of remained protein
Extraction time (min) Oil amount(g) Total oil amount(g) Oil content of dry basis (%)
0 not detected 1.16 ± 0.0 92.8 ± 0.2
5 2.43 ± 0.3 1.6 ± 0.1 74.1 ± 0.5
10 2.87 ± 0.1 1.50 ± 0.1 76.1 ± 0.5
15 3.24 ± 0.4 1.21 ± 0.1 81.8 ± 0.2
20 3.27 ± 0.2 1.17 ± 0.2 83.9 ± 0.4
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Values are presented as mean value ± SD (n = 3). Table 6 The effect of centrifugal force on emulsification. Centrifugal force (g) Emulsification rate (%)
120 6.0 ± 0.1
490 3.2 ± 0.0
1100 2.1 ± 0.1
1960 1.8 ± 0.2
3070 1.6 ± 0.1
Values are presented as mean value ± SD (n = 3).
Fig. 1. Process route of oil extraction.
206
4420 0.8 ± 0.0
6020 1.1 ± 0.2
7870 0.9 ± 0.3
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3.3. The optimized process of oil extraction
protein to the emulsion, which total oil content is nearly 100%. And petroleum ether also had a good effect on demulsificatio. According to the results, the process of soybean oil extraction, that is, isolating oilenriched protein after the enzymatic hydrolysis of soy emulsion, hydrolyzing the isolated protein again, and extracting the oil with petroleum ether, is much easier to be industrialized than those reported in former articles.
The above results showed that the enzymatic hydrolysis of soy emulsion could promote oil release, but the amount of released oil was smaller. Proteins isolated from hydrolyzed soy emulsion could adsorb nearly 95% oil of the emulsion through low-speed centrifugation at isoelectric point. The introduction of organic extraction in vegetable oil could significantly enhance the oil transfer in the emulsion and promote oil release from proteins. Meanwhile, the problem of demulsification was solved preferably. Namely, soybean emulsion was hydrolyzed by protease, then oil-enriched protein was isolated through low-speed centrifugation near isoelectric point of protein for further hydrolysis, finally the oil was extracted by petroleum ether. The process route is as shown in Fig. 1. The technological process and conditions were determined as follows: 800 mL soybean emulsion was prepared by 100 g soybean. Then the pH of emulsion was adjusted to 7.0, then the emulsion was hydrolyzed by 1398 neutral protease (240 IU/g soybean) at 45 ℃ for 1.5 h, with stirring rate 300 rpm. After that, the pH of hydrolysate was adjusted to 4.5, and centrifuged at 1960 × g for 15 min, to isolate the oilenriched protein. 200 mL distilled water was added to the separated proteins, and they were mixed well to form an emulsion. Then protease (480 IU/g soybean) was added to the solution, which was hydrolyzed at pH 7.0 for 2 h. After enzymatic hydrolysis, pH of the solution was adjusted to 7.0. Each 200 mL emulsion was extracted by 120 mL petroleum ether at 30 ℃ for 15 min, and the organic phase was separated at 1960 × g for 15 min. Soybean oil was obtained through vacuum evaporation. Oil yield was 89.6 ± 0.2%. The acid value of extracted oil was 2.60 mg KOH/g oil and the impurity 0.22% according to the standard AOAC 3d 63 method (Association of Official Analytical Chemists, 1997). The above experimental results show that, oil recovery can be obviously increased after the separation of oil-rich protein and organic solvent extraction after enzymatic hydrolysis according to the process shown in Fig. 1. Meanwhile, the method of demulsification was simpler and more convenient than former literature. In this research, 1398 neutral protease was used in aqueous extraction of soybean oil, which total dosage was only 720 IU/g. And the centrifugal force for oil separation was 1960 × g. The economic cost is lower than the reported methods. Soybeans do not need pretreatment by high temperature and high pressure extrusion.Thus, the process is easier to be industrialized. At the monment, 77 wt.% of soy protein was retained in water after the first enzymatic hydrolysis, which was favorable for recycling as a food resource. The introduction of petroleum ether into enzyme-assisted aqueous extraction of soy oil not only promotes the oil yield, but also helps to demulsification. Compared with the methods of demulsification reported in literature, it is more suitable for industrial application. The influence of the coexistence of petroleum ether and water in oil extraction on oil quality and protein is obviously less than the traditional solvent extraction, which is similar to that of Li et al. (2014), but without ultrasonic treatment. This study explored a new method for the industrialization of the process of enzyme-assisted aqueous extraction of soy oil.
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4. Conclusion After aqueous extraction, the obtained soybean emulsion contains 95.5% of oil and 95.0% of protein of original soybean. From this study, we found that the cheap 1398 neutral protease can obviously promote oil release and oil transfer during enzymatic hydrolysis. Oil-enriched protein, which adsorbed 99.0% oil from emulsion, could be obtained with low-speed centrifugation near isoelectric point. Petroleum ether extraction enhances oil transfer from the emulsion to the organic phase, and further promotes the oil release from the internal of the hydrolyzed
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Study on lipids transfer in aqueous enzyme hydrolysis soybean protein and oil extraction process
T
Junqing Qian , Jun Tong, Yan Chen, Shen Yao, Hui Guo, Lanhua Yang ⁎
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, China
ARTICLE INFO
ABSTRACT
Keywords: Enzyme-assisted aqueous extraction Soybean oil Oil transfer Proteolysis Neutral protease Petroleum ether
Enzyme-assisted aqueous Extraction of soybean oil is a promising green and safe alternative to traditional process. However, it is quite difficult to realize industrial application as the process needs large dosage of special enzyme and high-speed separation. To overcome these problems, the quantitative transfer of oil release during the hydrolysis of soybean protein by protease was studied. The results showed that the 1398 neutral protease with the total amount of only 720 IU/g soybean could be used to hydrolyze the soy protein, and the oil yield reached up to 90% after separation by the centrifugal force of 1960 × g. The enzymatic hydrolysis of soybean protein was beneficial to release and separate oil from soybean, and the separated protein could enrich almost 99.0% of oil from hydrolyzed emulsion. At the moment, the emulsification could be eliminated during the process of aqueous extraction with petroleum ether.
1. Introduction Soybean is one of the main edible oil seeds in the world. It is not only used for oil products, but also for the manufacture of high-quality vegetable protein (Loman and Ju, 2017; Ebert et al., 2017). Traditionally, soybean was usually treated at a high temperature to achieve higher oil yield because of low oil content. However, the process of high temperature treatment leads to the serious denaturation of soybean protein, and also affects the quality of oil (Galloway, 1976; Lawhon et al., 1981; Farhadian et al., 2011; Bojanowska and Czerwiński, 2010; Orecchio et al., 2009). For the improvement of the quality of soybean oil and the utilization of soybean protein, the process of enzyme-assisted aqueous extraction has been widely studied. Rosenthala et al. (2001) selected the optimum conditions of aqueous enzymatic extraction of soybean oil by Response Surface Methodology. At the moment, they tested the individual effect of two different enzymes—protease and cellulase—on oil yield with the best process parameters. It was found that protease could significantly contribute to a higher oil yield, 58.7%. Rovaris et al. (2012) explored the relationship between four enzyme systems and soybean oil yield, while the oil yield was only 23%. The amount of enzyme (2.5 wt.%) was increased by Moura et al. (2010) for de-emulsifying of protein emulsion from soybean, while the result was unexpected. The oil yield was improved by adding n-hexane finally. It was reported that pretreatment may improve oil recovery. Jung and Mahfuz (2009) squeezed soybean emulsion at 100 °C and 500 MPa, then
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0.5 wt.% of protease Protex 7 L was added for aqueous extraction, which promoted a significant increase in oil recovery (90%). After evaluating different proteases’contribution to free oil, Protex 6L was selected by Tabtabaei and Diosady (2013) at a concentration of 2.5 wt. % for hydrolysis. Results showed that 91% of oil from the emulsions can be recovered effectively with gradient centrifugation (9000 × g). The above studies on pre-treatment methods such as high temperature and pressure, and the selection of optimal protease showed that almost 90% of soybean oil could enter into the emulsion. The above studies on pretreatment methods such as high temperature and pressure, and the selection of optimal protease showed that almost 90% of soybean oil could enter into the emulsion. Meanwhile, it is crucial to demulsify and separate oil-water two phases for obtaining edible grade soybean oil. Several destabilization methods have been used to release oil from the emulsion formed during enzyme-assisted aqueous extraction of soybean. Protex 50FP and Protex 6L were used for soybean hydrolysis, respectively by Jung et al. (2009) and Wu et al. (2009). Under the optimal condition, nearly 90% of oil entered into the emulsion. Then the emulsion was demulsified by adding salt and organic solvent. Finally, 88% of free oil recovery was obtained. The centrifugal force (10,000 × g) was increased by Towa et al. (2011) after the emulsion treated at 96℃ for the oil yield, and the final yield reached to 90%. Bell et al. (2013) found that soybean oil extracted by enzyme-assisted aqueous extraction has some advantages such as low peroxide value and acid value. A process combing ohmic heating and enzyme assisted
Corresponding author. E-mail address: [email protected] (J. Qian).
https://doi.org/10.1016/j.indcrop.2019.04.063 Received 19 August 2018; Received in revised form 20 April 2019; Accepted 28 April 2019 Available online 20 May 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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aqueous extraction process was investigated by Pare et al. (2014) to improve soy oil recovery, however, the maximum oil recovery (73%) was still lower than other reports. To maximize the free oil yield of soybean oil, ultrasound-assisted enzymatic extraction was developed by Li et al. (2014). A significant efficiency of demulsification was obtained, and the oil recovery reached up to 92.6%. It is the highest oil yield in enzyme assisted aqueous extraction of soybean oil so far. Mechanism of enzyme-assisted aqueous extraction of soybean oil intrigued Han et al. (2015). Although oil recovery was only 69%, they stilled used microscope to intuitively observe the processes of the cells destruction, oil transfer and demulsification during enzymatic hydrolysis of soybean emulsion. Selection of enzymes and demulsification during enzyme-assisted aqueous extraction of soybean oil were studied widly. However, as a vegetable oil extraction method, quantitative studies on oil transfer in enzymatic hydrolysis of soybean emulsion are rarely reported. Although microscopic observation can directly demonstrate the enzymatic hydrolysis process and tissue fragmentation of soybean, it is impossible to obtain the oil transfer quantitatively, which is the basis of extraction process. Some methods such as the selection of outstanding performance enzyme with a dosage up to 2.5 wt.% (Tabtabaei and Diosady, 2013), the increase of centrifugal force (9000 × g) for emulsion separation (Tabtabaei and Diosady, 2013), and the use of ethanol (73%) with ultrasonic treatment for demulsification (Li et al., 2014) helped release oil during the process of enzyme-assisted aqueous extraction. The most highest oil yield could reach up to 92.6%, nevertheless it is not only difficult to industrialize on a large-scale, but unbearable in terms of economic cost for preparation of edible soybean oil. It is necessary to explore a better method of demulsification, which employs an enzyme with suitable performance, lower price and dosage, low speed centrifugation for oil-rich emulsion separation. At the moment, the largescale industrialization and relatively high free oil recovery should be achieved. The quantitative transfer of released oil in the process of protease hydrolysis of soybean protein was studied to overcome above problems, and a process using lower dosage of cheap enzyme for soybean oil extraction was established in this paper. Results showed that the coexistence of petroleum ether and water was beneficial to oil transfer, and had a good effect on demulsification. Therefore, the hydrolysis of soybean protein using 1398 neutral protease with 240 IU/g soybean was explored for the first time to promote the oil diffusion into aqueous phase. The soybean protein with a centrifugal force of 1960 × g was separated by adjusting the pH value of the emulsion,which was enriched 44% oil. Then, oil-enriched soybean emulsion was hydrolyzed by 1398 neutral protease (480 IU/g) once again. Almost 99% of oil could enter into the organic phase after adding petroleum ether. Two phases water and organic were separated with a centrifugal force of 1960 × g, and the emulsification ratio was less than 2.0%. Petroleum ether was recovered by low-vacuum evaporator, and 89.6% of the total oil of soybean was obtained. The acid value of the oil was 2.60 mg KOH/g oil, and the impurity was 0.22%.
Preparative Ultracentrifuge and Hitachi CR20B2 High Speed Refrigerated Centrifuge were used. 2.2. Preparation of soybean aqueous extraction Soybeans (100 g) were crushed to pass through a 20-mesh standard sieve, then soaked in distilled water (400 mL, pH 9.0), and extracted at 50 ℃ for 1.5 h. Next, they were grinded after adding 2 times volume of water, and filtered with 100-mesh sieve. The filter residue was washed by 2 times volume of water. The filtrate was collected for further study. Under this condition, 95.5% (w/w) oil and 95.0% (w/w) protein of soybean entered into aqueous extract emulsion (Qian et al., 2010). 2.3. Research method of lipids transfer in soybean aqueous extract emulsion after enzymatic hydrolysis Soybean aqueous (200 mL) extract emulsion was hydrolyzed by 1398 neural protease at pH 7.0 and 45 ℃ water bath, with stirring rate 300 rpm. Followed by centrifuging at 18,000 × g for 15 min, the system was split two layers (solid phase and aqueous phase) from top to bottom. Followed by centrifuging at 18,000 × g for 15 min, the system was split two layers (solid phase and aqueous phase) from top to bottom. The oil released from soybean protein has a lower density than water, forming upper solid phase. Then the dry basis oil content and total oil of upper solid were determined. Comparing oil release under different conditions, including enzyme dosage and enzymatic time, the effect of enzymatic hydrolysis on the oil release from soybean protein was studied. After enzymatic hydrolysis, pH of the system was adjusted to the isoelectric point of soybean protein. Then it was centrifuged at 1400 × g (15 min) for the soybean protein separation. The dry basis oil content and total oil content of the separated protein were determined to investigate the absorption of separated protein to oil, and evaluate the oil transfer from protein during enzymatic hydrolysis. 2.4. Research method of lipids transfer of soybean emulsion in the presence of petroleum ether Soybean emulsion (200 mL) was hydrolyzed by 1398 neural protease at pH 7.0 and 45 ℃ water bath, with stirring rate 300 rpm. The pH of 200 mL above emulsion was adjusted to 4.5, then 120 mL petroleum ether was added into the solution to extract for 15 min at 30 ℃. The solution was centrifuged at 4420 × g (15 min) for the separation of organic phase from the system. The oil was obtained by evaporation petroleum ether. And the transfer of oil into petroleum ether was investigated. The pH of emulsion separated from petroleum ether was adjusted to 7.0, then the emulsion was centrifuged at 18,000 × g for 15 min. The dry basis oil content and total oil of the upper solid were determined. From this, the release of soybean oil could be compared with the emulsion without petroleum ether. 2.5. Statistical analysis
2. Materials and methods
All analysis was done in triplicate. Data were expressed as the mean ± standard deviation of three replicates. STATISTICA 17 (StatSoft, Tulsa, OK, USA) was applied to conduct the ANOVA test and compare the means of the oil yield at 95% confidence interval.
2.1. Materials Soybeans were purchased from local market (Zhejiang, China). The initial moisture content of soybeans was 12.4% mass (wet basis). The oil content of soybeans was 18.6% mass (dry basis) determined by Soxhlet n-hexane extraction (American Society for Testing and Materials, 2009). The protein content of soybean was 40.5% mass (dry basis). The 1398 Neutral Protease (edible grade) was provided by Wuxi Enzymes Preparation Factory (Jiangsu, China) and the enzyme activity was 120,000 IU/g. All other chemicals and reagents used in this study were analytical grade and commercially available. Beckman LB-55M
3. Results and discussion 3.1. Lipids transfer in soybean aqueous extract emulsion after enzymatic hydrolysis It has been reported that enzymatic hydrolysis is helpful for oil release (Kapchie et al., 2008; Yusoff et al., 2015). According the above 204
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Table 1 Comparison of emulsion release oil with different enzyme dosage. Enzyme dosage (IU/g soybean)
0
240
480
720
960
1200
Released oil
0.65 ± 0.0 56.7 ± 0.1
1.01 ± 0.1 94.4 ± 0.2
1.12 ± 0.0 92.6 ± 0.1
1.09 ± 0.4 92.4 ± 0.5
1.14 ± 0.2 91.9 ± 0.4
1.07 ± 0.2 92.2 ± 1.3
Total oil (g) Oil content of dry basis (%)
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Time of enzymatic hydrolysis is 1.5 h. Values are presented as mean value ± SD (n = 3).
centrifuged at different speed for 15 min, for the study of the effect of centrifuge speed on lipid transfer, results are shown in Table 4. Both results (Tables 3 and 4) suggest that the oil transfer was enhanced after enzymatic hydrolysis. A large number of released oil was adsorbed by proteins which were separated with low-speed centrifugation near isoelectric point, so oil content is greatly improved. The total mass of separated protein and the total content of oil reached to 38% and 94.5% (about 99.0% oil of emulsion) of original soybean oil content, respectively. So it is meaningful for enzyme-assisted aqueous extraction to isolate oil-enriched protein, and this type of phenomenon of lipids transfer in emulsion has never been reported.
paper, emulsion was centrifuged at 18,000 × g after enzymatic hydrolysis, released oil that was less dense to water floated to the top, and the oil binding with protein still remained in the emulsion. Therefore, the effect of enzymatic hydrolysis on oil release under different conditions can be evaluated, through determining the oil content of upper solid and total oil. In order to reduce the protein adsorption to oil, the pH of soybean emulsion was adjusted to 6.0 and 7.0, respectively, according to the isoelectric point (IP) of soybean protein. Then the emulsion was centrifuged for 15 min, and the oil content of upper solid were 84.0% and 93.9%, respectively. Results showed that soybean protein entrapped less oil at pH 7.0, leading to a more accurate evaluation. Finally, the condition of pH (7.0) was selected for further study. Oil release under different enzyme dosages (Table 1) and enzymatic hydrolysis time (Table 2) was determined (each experiment consumed 200 mL emulsion (which oil content is 4.44 g)). From Table 1, the amount of released oil after enzymatic hydrolysis was obviously higher than the solution without enzymatic hydrolysis. The amount of enzyme was up to 480 IU/g soybean, oil content was to be stable, which showed that enzymatic hydrolysis was beneficial to oil release. From both Tables 1 and 2, although enzymatic hydrolysis was helpful for oil release, a small amount of oil was only obtained, and the highest only reached at 26.1% (w/w). Compared with the traditional oil extractions, oil yield appeared to be much lower. In this study, highspeed centrifugation (18,000 × g) was used to separate oil. But with the enzyme dosage increasing, the amount of oil did not increase, and it seems that extending enzymatic hydrolysis time was meaningless. Above all, soybean oil still contained protein (about 7%) after highspeed centrifugation. Results revealed the reason for lower oil yield of enzyme-assisted aqueous extraction which was reported in other literature. Therefore, it was rather difficult to extract oil through enzymeassisted aqueous extraction from vegetable protein. The above experiments confirmed that enzymatic hydrolysis was helpful for oil release, while it was extremely difficult to separate the oil from protein completely. That is, the transfer of lipids in emulsion was enhanced, but the released oil was adsorbed by protein. It doesn’t seem there is a strong connection between oil transfer and oil release. Next, the solids (proteins) were separated from soybean emulsion after enzymatic hydrolysis with low-speed centrifugation, for the study of lipids transfer. According to Table 1, 200 mL emulsion was hydrolyzed in 45 ℃ for 1.5 h at following conditions: enzyme dosage 240 IU/g soybean; pH 7.0; stirring rate 300 rpm. Then, the pH of the solution was adjusted close to isoelectric point before centrifuging (1400 × g). Results of oil content of separated protein are shown in Table 3. From Table 3, we adjusted the pH of solution to 4.5, then it was
3.2. Lipids transfer in soybean emulsion in the coexistence of petroleum ether Enhancing the transfer of lipids in aqueous phase contributes to efficient oil extraction from soybean protein. In the following research, the common method of vegetable oil extraction was introduced, and the oil transfer during the petroleum ether extraction was studied. The oil transfer in the organic phase under different extraction time was investigated, and the oil release of remained protein was evaluated through calculating oil content of upper solid and total oil after centrifuging at 18,000 × g (each experiment consumed 200 mL emulsion). Results are shown in Table 5. From Table 5, we found that 73% oil of emulsion that was extracted by petroleum ether entered into the organic phase. And the number of oil release from remained protein was greater than that was shown in Tables 1 and 2 . The sum of the oil released from enzymatiac emulsion and remained protein nearly reached 100% of the total oil, which indicated that petroleum ether extraction in enzyme-assisted aqueous extraction can obviously promote the oil transfer in internal hydrolyzed protein. The structure of protein was destroyed through enzymatic hydrolysis, leading to a easier lipids transfer. Meanwhile, organic extraction makes it easie to transmit oil from internal protein to the surface. And the extension of extraction time also helped oil transfer outward. After 15 min at equilibrium, the whole oil could be released under high-speed centrifugation. It was a great breakthrough for enzyme-assisted aqueous extraction reported in literature. Better oil transfer has been achieved, while the problem of emulsification needs to be solved. Next, the effect of different centrifugal forces on emulsification after extracting for 15 min was studied and the results were shown in Table 6. Emulsification rate could be controlled less than 2.0%, under the condition of centrifuging at 1960 × g for 15 min. Compared with the methods reported in former literature of demulsificasion, it was easier to be realized.
Table 2 Comparison of emulsion release oil different time of enzymatic hydrolysis. Time of enzymatic hydrolysis (h)
0
0.5
1.0
1.5
2.0
Released oil
0.65 ± 0.0 56.7 ± 0.1
0.75 ± 0.0 93.8 ± 0.1
1.03 ± 0.2 91.2 ± 0.3
1.16 ± 0.1 92.8 ± 0.2
1.14 ± 0.2 92.7 ± 0.4
Total oil (g) Oil content of dry basis (%)
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Enzyme dosage is 480 IU/g soybean. Values are presented as mean value ± SD (n = 3). 205
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Table 3 Oil adsorption by separated protein after hydrolyzed at different pH. pH
3.5
4.0
4.5
5.0
5.5
Dry weight of protein (g) Oil content (%) Percentage of total oil (%)
8.67 ± 0.2 41.9 ± 0.1 81.8 ± 0.2
9.26 ± 0.3 43.7 ± 0.2 91.1 ± 0.0
9.49 ± 0.1 43.8 ± 0.3 93.6 ± 0.1
8.95 ± 0.1 43.2 ± 0.2 87.1 ± 0.1
8.63 ± 0.2 41.5 ± 0.4 80.7 ± 0.3
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Values are presented as mean value ± SD (n = 3). Table 4 Oil adsorption by protein separated at different centrifugal force. Centrifugal force (g)
1100
1400
1960
3070
4420
Dry weight of protein (g) Oil content (%) Percentage of total oil (%)
8.56 ± 0.3 42.3 ± 0.2 81.5 ± 0.0
9.47 ± 0.2 43.9 ± 0.1 93.6 ± 0.2
9.51 ± 0.2 44.1 ± 0.1 94.5 ± 0.1
9.52 ± 0.1 43.2 ± 0.3 92.6 ± 0.2
9.51 ± 0.1 40.6 ± 0.3 87.0 ± 0.0
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Values are presented as mean value ± SD (n = 3). Table 5 Oil transfer of extraction phase and oil release of remained protein with different time of extraction. Extraction phase Oil release of remained protein
Extraction time (min) Oil amount(g) Total oil amount(g) Oil content of dry basis (%)
0 not detected 1.16 ± 0.0 92.8 ± 0.2
5 2.43 ± 0.3 1.6 ± 0.1 74.1 ± 0.5
10 2.87 ± 0.1 1.50 ± 0.1 76.1 ± 0.5
15 3.24 ± 0.4 1.21 ± 0.1 81.8 ± 0.2
20 3.27 ± 0.2 1.17 ± 0.2 83.9 ± 0.4
The oil content of soybeans was 18.6% mass (dry basis) and the protein content of soybean was 40.5% mass (dry basis). Values are presented as mean value ± SD (n = 3). Table 6 The effect of centrifugal force on emulsification. Centrifugal force (g) Emulsification rate (%)
120 6.0 ± 0.1
490 3.2 ± 0.0
1100 2.1 ± 0.1
1960 1.8 ± 0.2
3070 1.6 ± 0.1
Values are presented as mean value ± SD (n = 3).
Fig. 1. Process route of oil extraction.
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4420 0.8 ± 0.0
6020 1.1 ± 0.2
7870 0.9 ± 0.3
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3.3. The optimized process of oil extraction
protein to the emulsion, which total oil content is nearly 100%. And petroleum ether also had a good effect on demulsificatio. According to the results, the process of soybean oil extraction, that is, isolating oilenriched protein after the enzymatic hydrolysis of soy emulsion, hydrolyzing the isolated protein again, and extracting the oil with petroleum ether, is much easier to be industrialized than those reported in former articles.
The above results showed that the enzymatic hydrolysis of soy emulsion could promote oil release, but the amount of released oil was smaller. Proteins isolated from hydrolyzed soy emulsion could adsorb nearly 95% oil of the emulsion through low-speed centrifugation at isoelectric point. The introduction of organic extraction in vegetable oil could significantly enhance the oil transfer in the emulsion and promote oil release from proteins. Meanwhile, the problem of demulsification was solved preferably. Namely, soybean emulsion was hydrolyzed by protease, then oil-enriched protein was isolated through low-speed centrifugation near isoelectric point of protein for further hydrolysis, finally the oil was extracted by petroleum ether. The process route is as shown in Fig. 1. The technological process and conditions were determined as follows: 800 mL soybean emulsion was prepared by 100 g soybean. Then the pH of emulsion was adjusted to 7.0, then the emulsion was hydrolyzed by 1398 neutral protease (240 IU/g soybean) at 45 ℃ for 1.5 h, with stirring rate 300 rpm. After that, the pH of hydrolysate was adjusted to 4.5, and centrifuged at 1960 × g for 15 min, to isolate the oilenriched protein. 200 mL distilled water was added to the separated proteins, and they were mixed well to form an emulsion. Then protease (480 IU/g soybean) was added to the solution, which was hydrolyzed at pH 7.0 for 2 h. After enzymatic hydrolysis, pH of the solution was adjusted to 7.0. Each 200 mL emulsion was extracted by 120 mL petroleum ether at 30 ℃ for 15 min, and the organic phase was separated at 1960 × g for 15 min. Soybean oil was obtained through vacuum evaporation. Oil yield was 89.6 ± 0.2%. The acid value of extracted oil was 2.60 mg KOH/g oil and the impurity 0.22% according to the standard AOAC 3d 63 method (Association of Official Analytical Chemists, 1997). The above experimental results show that, oil recovery can be obviously increased after the separation of oil-rich protein and organic solvent extraction after enzymatic hydrolysis according to the process shown in Fig. 1. Meanwhile, the method of demulsification was simpler and more convenient than former literature. In this research, 1398 neutral protease was used in aqueous extraction of soybean oil, which total dosage was only 720 IU/g. And the centrifugal force for oil separation was 1960 × g. The economic cost is lower than the reported methods. Soybeans do not need pretreatment by high temperature and high pressure extrusion.Thus, the process is easier to be industrialized. At the monment, 77 wt.% of soy protein was retained in water after the first enzymatic hydrolysis, which was favorable for recycling as a food resource. The introduction of petroleum ether into enzyme-assisted aqueous extraction of soy oil not only promotes the oil yield, but also helps to demulsification. Compared with the methods of demulsification reported in literature, it is more suitable for industrial application. The influence of the coexistence of petroleum ether and water in oil extraction on oil quality and protein is obviously less than the traditional solvent extraction, which is similar to that of Li et al. (2014), but without ultrasonic treatment. This study explored a new method for the industrialization of the process of enzyme-assisted aqueous extraction of soy oil.
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4. Conclusion After aqueous extraction, the obtained soybean emulsion contains 95.5% of oil and 95.0% of protein of original soybean. From this study, we found that the cheap 1398 neutral protease can obviously promote oil release and oil transfer during enzymatic hydrolysis. Oil-enriched protein, which adsorbed 99.0% oil from emulsion, could be obtained with low-speed centrifugation near isoelectric point. Petroleum ether extraction enhances oil transfer from the emulsion to the organic phase, and further promotes the oil release from the internal of the hydrolyzed
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