Simultaneous Recovery of High Quality Black Sesame Oil and Defatted Meal by a New Aqueous Method: Optimization and Comparison with Other Methods

defatted （ ） Although cold Abstract: A method able to simultaneously obtain oil and defatted meal (rich in proteins) with high quality is preferable to others for processing black sesame seeds, which should also be green, healthy, highly efficient and sustainable. Methods including solvent extraction and hot-pressing currently available for the commercial production of oils are not able to meet all criteria just mentioned above. Therefore, development of new aqueous method of extracting black sesame oil has been promoted. In our study, we developed a new aqueous method using 1.95:10 aqueous salt solution-to-ground black sesame seed ratio which simultaneously recovered 96.54% black sesame oils and defatted meal with only 3.89% residual oils and 50.1% proteins (on dry weight basis). The oil produced had low acid value at 0.43 mgKOH/kg and peroxide value 3.37 mmol/kg and good other quality indexes. We found that proper amount of water added was essential for efficiently recover black sesame oils while other factors including temperature and time of baking raw materials to deactivate lipase activity, pore size of the sieve for ground black sesame seeds to pass through, addition of salt as well as temperature and time of agitating significantly affected the recovery efficiency. As compared with other methods, the new aqueous method had higher oil recovery rate or quality and was more environmentally friendly. No waste water was discharged during separation of oils. The experimental data can be applied to guide the design and manufacture of production line of black sesame oilseeds on a pilot or commercial scale.

solvent extraction not environmentally friendly and hot pressing poor quality and low value defatted meal has been traditionally applied to the commercially simultaneous production of high quality black sesame oil and defatted meal rich in proteins on a large scale and on a small scale, respectively, they have disadvantages 19,20 . Other methods such as supercritical fluid extraction method 21 and reverse micelle method 22 have not been applied to the commercial production of black sesame oils. Therefore, aqueous extraction with and without enzymatic assistance of black sesame oil using large quantities of water has been studied recently 23 26 , which still has disadvantages e.g. formation of serious emulsion, low free oil yield, discharge of large quantities of waste water, high cost of drying de-oiled residue or using enzyme, loss of significant amount of valuable compounds with waste water 27 32 .
The aim of this study was to develop a new aqueous extraction method of oils using black sesame seeds as studying materials and compare this method with hot pressing, traditional aqueous method assisted by enzymes and hexane extraction. In opposite to traditional aqueous extraction with and without enzymatic assistance of black sesame oil using large quantities of water free water to solubilize or disperse hydrophilic compounds and produce oil/water emulsion, the new aqueous extraction method of oils using small quantities of water to promote the aggregation of oilseed solids and water fully absorbed with the avoidance of solubilizing or dispersing hydrophilic compounds so that free oil is released. Study on the processing of black sesame seeds by the new aqueous method has never been reported before.

Materials
Black sesame seeds were obtained from Yonghui Supermarket, Beibei District, Chongqing, China. Distilled water was used. Sodium chloride salt which was food grade and all other chemicals or reagents used for chemical analysis were of analytical grade.
2.2 Optimization of new aqueous extraction method 2.2.1 General procedure used for all optimization experiments The schematic of separating oils by the new aqueous method is shown in Fig. 1. The black sesame seeds with peel were baked at a certain temperature variable studied for a certain time variable studied . The baked kernels were cooled and ground to pass through a sieve having a certain pore size variable studied to obtain ground black sesame seeds GBSS . A certain amount variable studied of aqueous salt solution with a certain concentration variable studied was added to 10 g GBSS in a 20 mL centrifuge tube. Then, the mixture of the salt solution and the GBSS was agitated for a certain time variable studied at a certain temperature variable studied by using a stainless steel rod. Finally, free oils were collected by centrifugation three times at 4000 r/min for 30 min followed by cold screw pressing once the presser made by Ai Bang Agricultural and Horticultural Machinery Plant, China and the defatted black sesame seed meal DBSSM was quantitatively collected and dried at 60 .

Optimization by single factor experiments
By following the procedure just mentioned above in General procedure used for all optimization experiments , the effect of several operating parameters on final oil yield FOY was investigated. All parameters investigated are summarized in Table 1 in details. The residual oil content in the DBSSM was determined by Soxhlet extraction. The oil extraction rate OER was calculated by using the following formula: OER X 1 X 2 X 1 100 1 In the formula, X 1 g represents the amount of crude oils in GBSS 10 g crude oil fraction in GBSS water free while X 2 g represents the amount of crude oils in the DBSSM amount of DBSSM g, water free its residual oil fraction . For new aqueous method, OER equals to FOY.
All experiments were conducted in triplicates.

Further optimization by response surface method
Response surface experimentation was conducted to check whether the new aqueous method of extracting sesame seed oil established by the single factor experimentation was the best or not, further optimize operating conditions and analyze the cross effect of multi-factors. By centering on the optimum experimental conditions established by the single factor experimentation, Box-Benhnken s central combined experimental design was carried out. Four factors including concentration of salt solution A , amount of salt solution added B , agitation temperature C , and agitation time D were selected and assigned to three different levels for performing response surface experiments.

Extraction of oils by solvent
The black sesame seed kernels were baked to the constant weight and crushed to pass a sieve with 154 μm pore size. The oil in the crushed kernel was extracted for 6 h by n-hexane using a Soxhlet extraction apparatus see Fig. 1 for the schematic of extraction procedure . After n-hexane was completely removed by rotary evaporation, the oil was vacuum-dried to the constant weight at 50 . The method optimized by Ma et al. 33 was followed to refine the crude oil extracted by n-hexane. Residual oil content in the DBSSM quantitatively collected and dried at 60 was analyzed according to Chinese National Standard Method GB 5009.6-2016 Chinese National Standards 34 . OER was determined according to the same method shown in Optimization of new aqueous extraction method .

FOY
was calculated by: total refined oil obtained g /total crude oil in the black sesame seed kernels g 100. 2 2.4 Production of oils by pressing at high temperature The extraction of black sesame oils by high temperature pressing was also conducted for comparison. BSS with peel were baked at 115 for 1 min and then pressed by the screw presser made by Ai Bang Agricultural and Horticultural Machinery Plant, China with the initial temperature of squeezing chamber at 100 . The DBSSM was quantitatively collected and dried at 60 for analysis of its residual oil content. OER and FOY were obtained according to the same method described in Extraction of oils by solvent .

Analytical methods
Crude oil content, acid value, peroxide value, smell and taste, solvent content, and transparency were analyzed according to Chinese National Standard Methods GB 5009.6-2016, GB 5009.229-2016, GB 5009.227-2016, GB/T 5525-2008, and GB/T 5009.37, respectively Chinese National Standards 34 . Color was analyzed by using a colorimeter HanterLab UltraScan Pro. USA 35 . Iodine value of oils was determined by fourier transform infrared spectroscopy developed by Che Man et al. 36 . Sesamin, sesamolin, sesaminol and sesamol were estimated by HPLC method reported by Young Kim et al. 37 .

Statistics analysis
The experimental data were analyzed using one-way analysis of variance. The significant difference between the pair data was estimated by Student s t-test. The t-value was calculated using Microsoft Office Excel 2010. Response surface analysis was carried out by using Design Expert 8.0.6 software. Multifactorial regression equation was established by using SAS V8.0.

Results and Discussion
The crude oil content of GBSS was 53.89 dry weight basis . All experiments used this sample as a studying material. FOY and OER were calculated on the basis of this oil content.

Optimization by single factor experimentation
The effect of added water on FOY is shown in Fig. 2. As shown in the figure, FOY increased significantly when the amount of water added increased from 0.00 to 1.40 mL. FOY was only ca. 5 when no water was added. Therefore, water is essential for the recovery of black sesame seed oils. However, FOY significantly decreased as the addition of water increased to be 1.50 mL and continuously reduced with further increases in water addition. In all dosages of water, 1.40 mL gave the maximum FOY 88.04 . Quantum theory considers that water has two proton acceptors and donors which are able to form hydrogen bonds with hydrophilic compounds having proton acceptors or donors. This can result in aggregation of the hydrophilic compounds and water in GBSS and subsequently the release of free oils. However, too much water may solubilize some hydrophilic compounds such as free fatty acids, phospholipids and phenolic compounds and subsequently increases their mobility. This can enhance the emulsification of oils and therefore reduce the FOY. Recovery of ca. 88 oils from BSS can be very meaningful for the application of defatted meal in the food industry since no other chemical is added during oil extraction and the preparation of some foods needs an appropriate amount of oils. For example, the defatted meal may be a good material for making black sesame paste a popular snack food in China and biscuits.
The effect of baking time at 110 on the FOY is shown in Fig. 2. As shown in the figure, FOY increased significantly when baking time increased to 1 min. However, the FOY was significantly reduced when baking time was longer than 1 min. This indicates that baking sesame seeds for 1 min is essential for obtaining a high FOY. The reason for this result may be that baking for 1 min may be long enough to completely deactivate lipase so that the losses of neutral fat during the aqueous extraction is reduced or ef-ficiently remove sufficient amount of water so that emulsification during the grinding process is avoided. However, baking sesame seeds for too long e.g. up to 2 min or longer may denature proteins, which may increase their oil-binding ability and thus reduce FOY. Rice proteins were reported to have a similar behavior 38 . Figure 2 shows the effect of baking temperature on FOY when baking time was fixed to be 1 min. As shown in the figure, FOY increased significantly as the baking temperature increased from 90 to 110 . However, when baking temperature increased to be 115 or higher, FOY significantly decreased. The reason for obtaining this result may be similar to that just described above in the first paragraph of this section.
The effect of agitation time on the FOY is shown in Fig.  2. As shown in the figure, FOY increased significantly when agitation time increased from 20 to 30 min. However, further prolonged agitation time significantly decreased the FOY. The reason for obtaining this result may be similar to that just described in the first paragraph of this section and the paragraph just followed.
The influence of agitation temperature on the FOY is shown in Fig. 2. As shown in the figure, FOY gradually increased when agitation temperature elevated from 25 to 60 . FOY increased dramatically to the maximum value when agitation temperature increased from 60 to 65 . However, the further increase in agitation temperature from 65 to 70 greatly reduced FOY. These results indicate that too high and too low agitation temperatures do not favor the efficient separation of oils from the solid particles of GBSS. The reason for these results may be as follows. An increase in temperature can decrease the viscosity of oils. This is advantageous for separating oils because the reduced viscosity at higher temperatures facilitates the coalescence of oils and their escape from the surface of solid particles. Furthermore, the mobility of oils and hydrophilic compounds may be improved by proper high temperatures. The more intense molecular motion of these compounds can promote the coalescence of oils and aggregation of hydrophilic compounds with water, respectively. The reason reducing the FOY may be similar to that described above in the first paragraph of this section.
The effect of particle size of GBSS on the FOY is shown in Fig. 2. As shown in the figure, it is obvious that the FOY dramatically increased when the particle size of GBSS decreased from 180 to 154 μm. Grinding GBSS to a certain extent can break its oil bodies and cell walls which is essential for the extraction of oils. Furthermore, decreases in the solid particle size of GBSS to a certain extent can increase its contact surface with water, which facilitates the aggregation of hydrophilic compounds and water, so that the release of oils is promoted. However, FOY significantly decreased as the particle size of GBSS decreased to be smaller than 154 μm. Too fine particles have large surface areas which may strongly absorb or emulsify oils so that FOY can be reduced. This may be supported by that oil/ water emulsion can be stabilized by fine food particles 39 . Therefore, grinding GBSS to pass through a sieve with a pore size of 154 μm was considered to be an appropriate choice for efficiently extracting black sesame oils.
The effect of concentration and amount of aqueous salt solution on FOY is shown in Fig. 2. With respect to studies on effect of salt concentration, FOY gradually increased as the concentration of aqueous salt solution increased from 4.76 to 6.43 w/w . However, FOY dramatically decreased when the concentration of aqueous salt solution further increased to be higher than 6.43 w/w . With respect to studies on effect of amount of aqueous salt solution added, FOY gradually increased when aqueous salt solution increased from 1.40 to 1.90 mL. However, FOY dramatically decreased when the amount of aqueous salt solution further increased to be higher than 1.90 mL. In all experiments, 1.90 mL aqueous salt solution with 6.43 w/w gave the maximum FOY 96.02 which was significantly higher than that 88.04 obtained by using pure water.
The mechanism of an increase in FOY by adding salt to water may be associated with changes in the surface tension and surface charge of protein or carbohydrate, which increases the polarity of the hydrophilic group. This effect can increase the non-covalent interaction of the polymer with water by the action of ionic bonds, and then extrudes the oil from the aggregated polymer. The addition of salt can also increase the water density and therefore increase the density difference between the aqueous sediment and the supernatant oil. On the other hand, too many salts may inhibit the aggregation of hydrophilic groups via hydrogen bonds by competing with them for water and subsequently decreases the production of free oil. Furthermore, an increase in sodium chloride level in the aqueous solution may enhance the hydrophobic interaction the driving force of the polymer chains adsorbed at the wateroil interface which involves in the interaction of the alkanes in the oil phase with polymer chains.
The single factor experimentation established new aqueous procedures for efficiently extracting black sesame oils. BSS with peel were baked at 115 for 1 min. The baked BSS were ground to pass through a sieve having 154 µm pore size to obtain GBSS. Aqueous salt solution 1.90 mL; 6.43 w/w sodium chloride in water was added to 10 g GBSS in a 20 mL centrifuge tube. Then, the mixture of the aqueous salt solution and the GBSS was agitated for 30 min at 65 by using a stainless steel rod. Finally, free oils were collected by centrifugation three times at 4000 r/min for 30 min followed by cold screw pressing once the presser made by Ai Bang Agricultural and Horticultural Machinery Plant, China and the DBSSM was quantitatively collected and dried at 60 . By using this method, FOY was as high as 96.02 . Extraction of black sesame oils by pure water were also found to be meaningful in terms of extracting oils with reasonable FOY and producing defatted meal with appropriate amount of residual oils applicable to the food industry.

Further optimization by response surface method
The experimental results indicated that four factors significantly affected FOY, which include concentration of aqueous salt solution, amount of aqueous salt solution added, and agitation temperature and time. Experimental results and design are shown in Table 2 In the formula, A represents concentration of aqueous salt solution, B represents amount of aqueous salt solution added, C represents agitation temperature, D represents agitation time. The results of significance test of this regression model for predicting FOY established were as the following: 1, the model F-value of 956.67 implied that the model was significant while there was only a 0.01 chance that a Model F-Value this large could occur due to noise; 2, values of Prob F less than 0.0500 indicated that model terms including A, B, AB, A 2 , B 2 and C 2 were significant; 3, the Lack of Fit F-value of 0.33 implied that the Lack of Fit was not significant relative to the pure error while there was a 93.01 chance that a Lack of Fit F-value this large could occur due to noise. Non-significant lack of fit was good --we wanted the model to fit.
The optimum combination of operating experimental conditions for giving the highest FOY 96.54 among all combinations studied was as the following: A 6.79 w/w; 0.95 g sodium chloride in 14 g aqueous salt solution , B 1.95 mL, C 65 , and D 30 min. It should be noted that 16 operating condition combinations out of 29 obtained FOY 96 while 23, 28, or 29 operating condition combinations obtained FOY 95 , 93 or 92 , respectively. All FOYs of higher than 96 were not significantly dif- ferent p 0.05 so that all operating condition combinations involved should be feasible for efficiently extracting BSS oils, but for cost control, an operating condition combination with smaller amount of salt and water, lower agitation temperature and shorter agitation time may be preferable.
It should also be noted that all FOYs of higher than 96 obtained by response surface experimentation were not significantly different from that 96.02 obtained by single factor experimentation p 0.05 . Response surface method also found that the cross  Single factor experimentation and response surface method found 1 and 26 operating condition combinations which recovered more than 96 black sesame oils, respectively. FOY of 27 operating combinations were not significantly different p 0.05 . All these operational condition combinations were found to be practically meaningful in terms of efficiently recovering black sesame oils. The most critical factor for obtaining high FOY was found to be 1.90 or 1.95:10 aqueous salt solution-to-GBSS ratio while other factors significantly affected FOY.
3.2 Comparing oil recovery rate and the quality of oil and defatted meal obtained by new aqueous method established and other methods Table 3 indicates the comparison of final oil yield and quality of oils and defatted meal obtained by the new aqueous method established in this study and other methods as compared with Chinese national standard. Although the new aqueous method developed in this study had slightly lower OER, it had comparable FOY i.e. oil recovery rate as compared with solvent extraction. The reason for this may be that the oil extracted by solvents cannot be consumed directly and should be refined The refining process of crude oil can cause significant losses of neutral oils 20 . In addition, as compared with the enzymeassisted water extraction method, the method developed in this study obtained significantly higher FOY though it may not result in a higher OER. As compared with hot pressing, new aqueous method developed in this study had significantly higher OER and FOY. Even, FOY obtained by using pure water was comparable to that obtained by hot pressing.
Proper acid value AV and peroxide value PV of oil are mandatorily required by national standard. The oil recovered by new aqueous method developed in this study had acid value AV and peroxide value PV better than that required by the Chinese National Standard CNS for the first class edible sesame oils. The oil recovered by solvent extraction had AV, PV and solvent content higher than that required by CNS so that it needs to be refined for edible purpose. The oil recovered by hot pressing had higher AV and better PV as compared with that required by CNS. Relevant data of the oil recovered by enzyme-assisted aqueous method using large quantities of water solution was not found. Iodine value, sesamin, sesamolin, sesamol and sesa- minol of crude oils extracted by the new aqueous method, solvent extraction and hot pressing were not significantly different. However, inevitably refining the crude oils extracted by solvent may cause significant losses of sesamin, sesamolin, sesamol and sesaminol. The oil produced by all methods compared was clear and transparent while it had the inherent smell and taste of sesame oil with no adverse smell. Hot pressing produced oil with stronger fragrance as compared to other methods. However, new aqueous method can also produce oil with strong fragrance by baking BSS for an appropriate long time if production of high quality of DBSSM is not attempted or required.
It was found that the color L* 65.56, a* 13.27, b* 103.07 of the oil produced by new aqueous method established in this study was lighter than that L* 31.08, a* 2.19, b* 5.60 obtained by solvent extraction, while that L* 4.03, a* 21.74, b* 6.94 of the oil produced by high temperature pressing was the deepest. The oil obtained by the method developed in this study had more light yellow color than the oil produced by solvent extraction and hot pressing.
The DBSSM produced by new aqueous method, solvent extraction or hot pressing was black like the original black sesame seeds. New aqueous method produced DBSSM with only about 28 water so that its drying can be easy. It produced DBSSM with slightly higher and significantly lower oil content as compared with solvent extraction and hot pressing, respectively. Furthermore, the protein content of the DBSSM produced by this advanced aqueous method or solvent extraction was 50.1 or 55.3 , respectively. Slightly higher residual oil content and small amount of salt added should be responsible for the lower protein content in the DBSSM produced by new aqueous method. Because the extraction of oils by new aqueous method was carried out at a low temperature 65 , nutritional and other functional compounds having hydrophilic groups such as B-vitamins, flavonoids or other polyphenols and phospholipids should not be damaged and remain in the DBSSM while proteins in it should not be denatured so that it can be a good material for producing protein isolate. The DBSSM may be directly used as a food material since whole sesame seeds with peel is edible. Their dark color should not be an obstacle of their application in foods since black sesame seeds as food products are well accepted by consumers. However, the DBSSM obtained by traditional high temperature pressing can only be used as feeding materials. This means that the new aqueous method developed in this study can also produce a high quality DBSSM in addition to oils from black sesame seeds.

Analysis of environmental effect
New aqueous method developed in this study did not produce waste water during extraction of oils. Conversely, enzyme-assisted aqueous method discharged large quantities of waste water.
New aqueous method did not release harmful substances during production of oils and defatted meal. It is well Having the inherent smell and taste of sesame oil, no adverse smell; c C,T-Clarify, transparent; d The highest extraction yield of sesame oil by enzyme-assisted aqueous method reported in the literature 24) .
Optimizing New Aqueous Method of Extracting Black Sesame Oils and Comparing with Other Methods known that solvent extraction of oil can emit harmful gas into atmosphere.
New aqueous method utilized all black sesame seeds. On the other hand, hot pressing produced low value defatted meal which should not be suitable for being applied to the food industry. This means that less protein sources, etc. need to be produced for meeting human s demand if new aqueous method is employed for extracting oils. Increases in production of protein sources, etc. should elevate the burden of environment.
The analysis described in the above paragraphs indicates that new aqueous method is a green technology. It is superior to other methods in term of protecting environment.

Conclusions
It was concluded that the addition of water or aqueous solution was essential for the separation of black sesame seed oils while other conditions including baking temperature or time, salt addition, sieve pore size that GBSS passed and agitation time or temperature significantly affected FOY. The new aqueous method eventually developed in this study recovered more than 96 of black sesame oil. Its FOY was comparable to that of solvent extraction and higher than that of enzyme-assisted aqueous method and hot pressing. It produced oil and defatted meal with higher quality as compared with solvent extraction and hot pressing. It is more environmentally friendly as compared with other methods. The new aqueous method should have the potential of becoming the major method of producing black sesame oils on a commercial scale in future.