Only one process condition varied with all others fixed and its level producing the maximum ORR was selected for the subsequent experiment. AAM procedures for investigation into the effect of baking time on ORR were as follows: “Raw PSKs were baked for 0, 1, 2, 3, 4, or 5 min, respectively, at 105 ºC, peeled, crushed and ground to pass through a sieve with 105 µm pore size producing ground PSK (GPSK). Water (1.50 ml) was added to a 20 ml centrifugal tube with 10.00 g GPSK. After stirring at room temperature (25 ºC) for 30 min, the oil was collected by centrifuging at 4000 r/min (1435 g) for 30 min thrice with 80-2 centrifuger (manufactured by Honghua Instrument Factory, China) and weighed. All residues from centrifugation were quantitatively collected and cold-pressed once with a presser (made by Ai Bang Agricultural and Horticultural Machinery Plant, China). All the de-oiled meal from the presser was quantitatively collected, dried, weighed and ground to pass through a sieve with 154 μm pore size for determining oil content according to the Soxhlet method. The extraction yield of oil (EYO) was calculated as follows:

In the formula, X₁ (g) represents the total oils of GPSK [ 10 g × crude oil fraction of GPSK] , while X₂ (g) represents the total oils of de-oiled meal (the amount of de-oiled meal (g, dry basis) × its oil fraction). For AAM, EYO is the same as ORR.

AAM procedures for the investigation into the effect of baking temperature on ORR. Raw PSKs were baked for 1 min at 100, 105, 110, 115, 120, or 125 ºC, peeled, crushed and ground to pass through a sieve of 105 µm pore size to produce ground PSK (GPSK). Water (1.50 ml) was added to a 20-ml centrifugal tube with 10.00 g GPSK. After stirring at room temperature (25 ºC) for 30 min, the rest of the procedure was the same as that described above in “AAM procedures for the investigation into the effect of baking time on ORR”.

AAM procedures for the investigation into the effect of water added on ORR. Raw PSKs were baked for 1 min at 110 ºC, peeled, crushed and ground to pass through a sieve of 105 µm pore size to produce ground PSK (GPSK). Water (0.00, 0.50, 1.00, 1.20, 1.40, 1.50, 1.60, 1.70, 1.80, or 1.90 ml) was added to a 20-ml centrifugal tube with 10.00 g GPSK. After stirring at room temperature (25 ºC) for 30 min, the rest of the procedure was carried out in the same manner as that described above in “AAM procedures for the investigation into the effect of baking time on ORR”.

AAM procedures for the investigation into the effect of sieve pore size on ORR. Raw PSKs were baked for 1 min at 110 ºC, peeled, crushed and ground to pass through sieves with 550, 270, 250, 212, 160, 150, 105, 75, or 58 µm pore sizes to produce ground PSK (GPSK). Water (1.60 ml) was added to a 20-ml centrifugal tube with 10.00 g GPSK. After stirring at room temperature (25 ºC) for 30 min, the rest of the procedure was the same as that described above in “AAM procedures for the investigation into the effect of baking time on ORR”.

AAM procedures for investigation into effect of stirring time on ORR. Raw PSKs were baked for 1 min at 110 ºC, peeled, crushed and ground to pass through a sieve with 150 µm pore size to produce ground PSK (GPSK). Water (1.60 ml) was added to a 20-ml centrifugal tube with 10.00 g GPSK. After stirring at room temperature (25 ºC) for 0, 15, 20, 25, 30, 35, 40, or 45 min, the rest of the procedure was carried out in the same manner as that described above in “AAM procedures for the investigation into the effect of baking time on ORR”.

AAM procedures for the investigation into effect of stirring temperature on ORR. Raw PSKs were baked for 1 min at 110 ºC, peeled, crushed and ground to pass through a sieve with 150 µm pore size to produce ground PSK (GPSK). Water (1.60) was added to a 20-ml centrifugal tube with 10.00 g GPSK. After stirring at 25, 30, 35, 40, 45, or 50 ºC for 30 min, the rest of the procedure was the same as that described above in “AAM procedures for the investigation into the effect of baking time on ORR”.

AAM procedures for the investigation into the effect of salt added on ORR . Raw PSKs were baked for 1 min at 110 ºC, peeled, crushed and ground to pass through a sieve with 150 µm pore size to produce ground PSK (GPSK). Water (1.60 ml) and salt (0.00, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, or 0.12) were added to a 20-ml centrifugal tube with 10.00 g GPSK. After stirring at 30 ºC for 30 min, the rest of the procedure was the same as that described above in “AAM procedures for the investigation into the effect of baking time on ORR”.

AAM procedures for the investigation into the effect of water added with the presence of 0.08 g salt on ORR. Raw PSKs were baked for 1 min at 110 ºC, peeled, crushed and ground to pass through a sieve with 150 µm pore size to produce ground PSK (GPSK). Water (0.00, 0.50, 1.00, 1.20, 1.40, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, or 1.80 ml) was added to a 20-ml centrifugal tube with 10.00 g GPSK plus 0.08 g salt. After stirring at 30 ºC for 30 min, the rest of the procedure was the same as that described above in “AAM procedures for the investigation into the effect of baking time on ORR”.

According to the results of the single factor experiments, sieve pore size (A), stirring time (B), water addition (C), and baking temperature (D) were selected for further optimization using the response surface method with all other process conditions fixed. For this purpose, Box-Benhnken’s central combined experimental design was employed.

Figure 2 shows the effect of water addition, baking time or temperature and sieve pore size on ORR. The effects of all these three factors were significant with different priorities.

ORR increased from 5.03 to 90.11% as water addition increased from 0 to 1.60 ml, but it continuously decreased when > 1.60 ml was added. The ORR of only 5.03% without water addition indicated that water addition was the major, essential and critical factor for efficiently separating oils. When water addition was at the level of 1.60 ml, solid particles absorbed all added water and aggregated together and the maximum release of free oils occurred. According to quantum chemistry theory, the aggregation of solid particles should be caused by the formation of hydrogen bonds because of water addition. However, the addition of too much water might cause the solubilization of some hydrophilic compounds, increase their movability, reduce the size of solid particles and therefore facilitate the formation of emulsion so that ORR decreased. The maximum ORR obtained was meaningful since it was higher than that obtained by EAAM (Hu and Zou, 2013). The amount (1.60 ml) of water added to 10.00 g raw materials (GPSK) for obtaining the maximum ORR did not cause any free water or result in waste water during the extraction of oils by AAM.

ORR increased from 79.01 to 86.02% as baking time increased from 0 to 1 min, but it continuously decreased for longer than 1 min while it increased from 80.31 to 89.02% as baking temperature increased from 100 to 110 ºC, although it continuously decreased at temperatures higher than 110 ºC. Proper heating treatment should deactivate lipase, which may reduce the losses in neutral oils during oil extraction and therefore elevate ORR. On the other hand, excessive heating (> 1 min and > 110 ºC) may cause the denaturation of proteins and increase their oil absorption capacity, which could therefore reduce ORR. The extent of variation in ORR caused by the variation in the heat treatment was much smaller than that caused by the variation in water addition, which means that heating treatment is a minor or an assistant factor.

ORR increased from 71.4 to 91.36% as sieve pore size reduced from 550 to 150 µm, but it continuously decreased when < 150 µm was used. Grinding PSKs to pass through a sieve with proper pore size should be necessary for fully breaking oil bodies and cell walls to release oils so that proper grinding increased ORR. However, excessive grinding might produce fine particles which are too small in size, which could increase the surface areas of the particles, elevate their ability to stabilize the emulsion (Dickinson, 2006) and therefore decrease ORR. This factor also had a smaller extent of variation in ORR compared to water addition, which should also be an assistant factor.

Figure 3 indicates the effect of stirring temperature or time, NaCl addition and water addition with the presence of 0.08 g NaCl on ORR. These three factors also significantly affected ORR to different extents.

ORR increased from 91.30 to 92.48% as stirring temperature increased from 25 to 30 ºC, but it continuously decreased at temperatures higher than 30 ºC. A reduction in oil viscosity and an increase in the intensity of molecular motion of oils and hydrophilic compounds might occur when stirring temperature is elevated, which might facilitate the aggregation of hydrophilic groups via hydrogen bonds as well as oil coalescence and release from the surface of aggregated solid particles so that ORR increased. On the other hand, too high a temperature (≥ 30 ºC) might lead to an increase in dispersity of surfactants, which might increase the formation of emulsion and thus reduce ORR. This factor also produced a smaller extent of variation in ORR compared to other factors, which should therefore be an assistant factor. ORR increased from 58.52 to 92.56% as stirring time increased from 0 to 30 min, but it continuously decreased after longer than 30 min. Proper stirring time should be necessary for the excess of water to hydrophilic groups of solid particles and the formation of hydrogen bonds. This might facilitate the aggregation of solid particles and the release of free oils from their surface. Excessive stirring might elevate the dispersity of surfactants which might cause emulsion and thus reduce ORR.

ORR increased from 92.40 to 94.07% as NaCl addition increased from 0.00 to 0.08 g, but it continuously decreased when > 0.08 g was added. With the presence of 0.08 g NaCl, ORR increased from 4.83 to 94.14% as water addition was increased from 0.00 to 1.60 ml, but it continuously decreased when > 1.60 ml was added. The addition of NaCl might increase the density or surface charge of aggregated solid particles and therefore facilitate the separation of oils. The addition of NaCl only increased ORR by 1.67% compared to the addition of pure water. Therefore, AAM without the addition of NaCL might also be meaningful in pumpkin seed processing practices.

Therefore, the optimal process conditions of AAM obtained by single factor experimentation were as follows: baking Pumpkin seeds at 110 °C for 1 min, crushing PSKs and grinding them to pass through a sieve with 150 μm pore size to obtain GPSK, adding 1.60 ml water and 0.08 g NaCl to 10.00 g GPSK, stirring for 30 min at 30 °C, centrifuging at 4000 r/min (1435 g) for 30 min thrice, and cold-pressing the residue from centrifugation once. The verification test proved that ORR was 94.08% under the optimal process conditions with the general procedures (see “2.2”) followed. Under the optimum conditions, centrifugation and the subsequent cold-pressing recovered 90.15% and 3.93% of total oils from GPSK, respectively. The AAM process conditions optimized in this study for obtaining the maximum ORR from PSKs were significantly different from those for processing other oilseeds previously published (Table 1).

The response surface method associated with sieve pore size (A), stirring time (B), water addition with the presence of NaCl (C) and baking temperature (D) did not improve ORR as compared to single factor experimentation. The results obtained by the response surface method are indicated in Table 2. However, Figures 4-6 show that AB, AC and AD had significant cross-effects on ORR. The second-order response surface equation (R² = 0.9943) was obtained to represent the final oil yield.

Single factor experimentation adequately optimized process conditions since the surface response method did not find different process conditions to give higher ORR. Adding the proper amount of water was essential and critical while baking seeds, grinding PSKs, stirring GPSK and adding NaCl significantly affected ORR. The optimal conditions established can provide useful scientific basis for process engineering purposes. According to the results of some (including experiments on baking, water or salt addition, sieve pore size and stirring) of this study, bakers (e.g. tunnel furnace) with the accurate control of heating temperature (110 °C) and time (1 min), grinders (e.g. roller-type) which can grind PSKs to pass through a sieve with 150 μm pore size (or destruct all oil bodies and cell walls without production of too fine solid particles) and stirrers (e.g. worm-type) with automatically quantitative addition of water or brine solution and feeding of GPSK are necessary for producing free oils and aggregated solid particles from PSKs on an industrial scale. Centrifugers and cold-pressers are good selections for collecting free oils and de-oiled meal.