Chapter 6: Particle Generation with Air-Assisted Sub-critical Water Extraction Technology
3.3. Results and Discussion
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samples with molecular weights ranging from 1.24 × 103 to 3.44 × 106 and a concentration of 0.05 %.
Particle structures of the powder microcapsules were evaluated by JEOL JSM-5200 model (Tokyo, Japan) scanning electron microscope. Powders were attached to SEM stubs using a 2-sided adhesive tape and left in desiccator. Then they were coated with a fine layer of gold through Sputter Coating Attachment of JEOL JFC- 1100 E (Jeol, Tokyo, Japan), in vacuumed evaporators before examination. For observation, a Scanning Electronic Microscopic JEOL JSM-5310LV (Jeol, Tokyo, Japan) working with a voltage of 15 kV. The microphotographs were carried out with a camera coupled to the microscopic. The samples were systematically observed with 1500 of magnification.
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Figure 3.7. Percent recovery of WSOC at various temperatures.
※The yield of water-soluble organic carbon (WSOC) (%)：
The weight of WSOC in filtrate / The weight of WSOC in material 100
Each experiment was conducted in triplicate with a relative standard deviation of 2-7 %. In the batch hydrothermal extraction of G. lucidum, samples were reacted in the tubular reactor using subcritical water for a specific contact time. It was found that although the recovery yield of total WSOC was affected by temperature; the lowest recovery was 7.52 % at 373 K. Fig. 3.7 shows temperature effect on WSOC recovery efficiency depending on time. The recovery was calculated using the reference data for the weight of WSOC in the samples by elemental analysis.
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Temperatures up the 473 K were gradually increased and showed positive effect on the recovery efficiency having highest values of 18.048 %, 19.37 % in individual temperatures for 373 K and 423 K respectively. After 473 K, the efficiency became lower again. The highest yield obtained at 473 K as 78.11 % and decreased to 61.59 % and 46.82 % for 523 K and 573 K respectively. The relation between the extraction efficiency and temperature can be neatly observed from Fig. 3.8.
Figure 3.8. Relation between extraction efficiency and reaction temperature.
Below the Table 3.2 indicates the results for batch-scale sub-critical water extraction of Ganoderma lucidum in terms of recovery efficiency for water soluble organic carbon amount (WSOC) (%) and recovery yield (mg WSOC/ g sample) as it is illustrated in Fig. 3.9 schematically in ppm units.
84 Temperature
(0C)
Time (min)
100 150 200 250 300
Selected Experi mental Conditi ons for TOC AnalyzeAmount of Sample Used for the Extracti on ( 0.05 g )
TOC Recovery (mg WSOC/ g sample) TOC Efficiency (%)
5 15.94 8.753 37.11 61.59 46.82
10 7.52 11.88 56.02 54.49 42.66
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Table 3.2. Efficiency and yield values for batch-scale sub-critical-water extraction.
20 11.33 11.95 66.06 47.83 36.74
30 8.9 14.08 66.49 45.43 39.52
40 11.55 15.18 67.25 46.73 34.28
50 18.048 15.88 69.25 43.50 40.21
60
5 10 20 30 40 50 60
10.418
66.9799 31.5990 47.6087 37.3978 48.5331 75.8377 43.7764
19.37
36.7801 49.9198 50.2139 59.1642 63.7864 66.7278 81.3927
78.11
155.9362 235.396 277.5841 279.391 282.5845 290.9885 328.2182
39.74
258.8012 228.967 200.9817 190.8969 196.3595 182.787 166.9875
38.39
196.7376 179.2573 154.3815 166.063 144.0446 168.9624 161.3148
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Figure 3.9. Recovery yield of WSOC at various temperatures in batch mode hydrothermal treatment.
To determine the extraction residues, we examined the water-insoluble organic carbon (WIOC) yield by gravimetric analysis. We found that solubility increased with increasing temperature up to 473 K, indicating a decrease in the amount of water-insoluble material. In contrast, for temperatures above 473 K, solubility began to decrease. The highest solubility (54.8 %) was obtained at 473 K as shown in Fig. 3.10.
The semi-continuous hydrothermal extraction of water-soluble polysaccharides was carried out by exposing the G. lucidum samples with a continuous flow of pure degassed water at subcritical temperatures with sufficient pressure to maintain the water
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in a liquid state. The temperature ranged from 373 to 473 K at a constant pressure and water flow rate of 10 MPa and 1 ml/min, respectively, for 130 min.
Figure 3.10. Water Insoluble Organic Carbon (WIOC) Extraction Efficiency (%).
※From Gravimetry;
The yield of water-insoluble fraction(%)＝Residue(dry-g)/Material(dry-g) 100
During the extraction process, the solubility of water soluble compounds sharply decreased after expanding the water using back pressure regulator where water was no more in sub-critical state. The extraction was carried out at operating conditions;
temperature ranging from 373 to 473 K at constant pressure and water flow rate of 10 MPa and 1 ml/min respectively for 130 min extraction time.
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As shown in Fig. 3.11, the extracted amount of total water-soluble organic carbon (WSOC) recovery including polysaccharides increased with an increase in temperature. The highest yield of WSOC extracted with sub critical water at 373, 423, and 473 K were 30.25, 74.72 and 241.05 mg WSOC g-1 of dry sample respectively. This temperature dependence of the yield is due to the increased solubility of water-soluble organic compounds in sub critical water as the water temperature increases.
2. 3. Analysis
2. 4. Results and Discussion
2. 3. 3. Temperature and Pressure Effects in Modified Supercritical Extraction
2. 4. Conclusion
Figure 3.11. Effect of temperature on the extraction curve, 10 MPa, 1 ml/min water flow rate.
Conventionally, Ganoderma lucidum extraction involved the methanol, ethanol, or hot water method [20]. Sye mentioned the conventional extraction methods. The yield of methanol extraction (1.35%) is better than that of two traditional methods.
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Contemporarily, the yield of methanol extraction was also higher (1.195 %). It, due to methanol, has the higher polarity and solubility. However, methanol is a toxic solvent that avoids remaining in food or medicine. Nevertheless, in this study, the yield of subcritical water extraction (5.36 %) is higher than that of the methanol method.
The total amount of WSOC recovery of the extracts containing polysaccharides increased with increasing temperature, with the highest yields of WSOC at 373, 423, and 473 K being 30.3, 74.7 and 241.1 mg WSOC/g dry sample, respectively. The highest cumulative extraction percentage yield (57.4 %) was achieved in extracts obtained at 473 K for a total duration of 130 min (Fig. 3.12). This temperature dependence is due to the increased solubility of water-soluble organic compounds in subcritical water as the water temperature increases. In other words, the solubility of water soluble compounds increased with increasing temperature.
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Figure 3.12. Effect of temperature on WSOC extraction for semi-continuous hydrothermal extraction.
The results suggest that extraction temperature greatly affects pressurized hot water extraction in terms of WSOC recovery (Fig. 3.13), and 473 K results in the most efficient extraction of WSOC in both the batch and semi-continuous methods. However, it is known that semi-continuous type extraction should be much better in recovery efficiency. In our case the batch process gave the higher results because of stabilized condition of water in the reactor and the rate of sample to water ratio. The rate difference was not so much as it is in semi-continuous type extraction, it was much more concentrated in batch process. On the other hand, it should be noted that pressure does not affect the performance of subcritical water extraction as long as it is high enough for the water to maintain the liquid state. However, the amount of water appears to positively affect extraction with a direct proportional relationship with efficiency, as shown in the plot against water to feed ratio in Fig. 3.14, a higher amount of water results in higher extraction recovery yield. Water to flow ratio was calculated by diving sample amount to consumed water during extraction. As it is seen from the figure, W/F ratio was same for batch-scale experiments because the reaction volume was constant;
there was no flow during the experiment. For semi-continuous type extraction where water flow rate was 1 ml/min, the ratio was calculated for each time fraction where the water consumption is equal to the time consumed.
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Figure 3.13. Comparison of extraction efficiencies for batch- and semi-continuous scale treatments, 473 K.
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Fig. 3.14. Effect of water to feed ratio on extraction at 473 K.
We also analyzed the molecular weight of the extracts from both methods by GPC. The molecular weight of the extracted polysaccharides was distributed around 500 g/gmol. Referring to the Gel Permeation Chromatography (GPC), it was done after both scales of experiments. For the estimation of average molecular weights of water-soluble organic compounds, calibration curve was formed using polystyrene standard kits including 7 portions having the molecular weights from 1.24*103 up to 3.44*106 taking the concentration as 0.05 % for all injected samples as it is illustrated in Figure 3.15 together with Table 3.3 that shows relevant calibration conditions.
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Figure. 3.15. GPC Calibration Curve and Analysis Conditions (same polystyrene standard concentrations- 0.05 %, 10 µl injection).
94 Table 3.3. GPC calibration analysis conditions.
All analysis conditions were same for the polystyrene standards and the sample material. By the end of analysis it was seen that the elution volume for the samples were too small comparing the values detected for polystyrene standards, changing around 9-10 ml for all extracted samples in both batch and semi-continuous scale extractions where the elution volume was at least 7.25 ml for the smallest size polystyrene standard.
This indicates that the long chained structure of the water soluble polysaccharides divided into very small portions in assistance of the high temperature effect so that the exact numerical detection could not be done referring to reference calibration curve.
Referring the representative GPC chromatogram for extracted samples (Fig. 3.16), it can be said that the molecular weights for the extracted water-soluble organic compounds, which are mainly composed of polysaccharides including mostly beta glucans, were very small. In addition to the case explained, it was also seen that the amount of extract was little bit increased with high temperature getting smaller in structural composition.
Analysis Conditions SAMPLE Polystyrene (Standard)
1 1240 7.25
2 3950 6.417
Column TSK gel GMHXL
(Tosoh Company)
3 13800 5.758
Eluent THF 4 31400 5.592
5 54100 5.408
Flow rate 1 mL/ min
6 202000 5.358
7 532000 5.267
Detector UV (254 nm)
8 1340000 5.125
9 2160000 5.033
Column temperature 25 oC
10
Molecular weight (MW)
3440000
Elution volume (mL)
4.942
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Figure 3.16. GPC Chromatogram.
Ganoderma extract powder was obtained by means of spray drying with Mini Spray dryer (YAMATO, ADL 310, YAMATO SCIENTIFIC CO., LTD., Tokyo- Japan).
96 (1)
Figure 3.17. Micrographs of particles of Ganoderma lucidum obtained by electronic microscopy after
hydrothermal water treatment, (1:
original sample, 2: 373 K, 3: 423 K, 4:
448 K, 5: 473 K).
(3)
(2)
(4)
(5)
The spray dryer operates concurrently and has a spray nozzle with an orifice of 1 mm in diameter. The experiments were performed at constant process conditions.
Scanning electronic microscopy (SEM) technique was used to visualize the structure of the powdered form of Ganoderma lucidum. Particle structures of the powder microcapsules were evaluated by JEOL JSM-5200 model (Tokyo, Japan) scanning electron microscope. Powders were attached to SEM stubs using a 2-sided adhesive tape
97
and left in desicator. Then they were coated with a fine layer of gold through Sputter Coating Attachment of JEOL JFC- 1100 E (Jeol, Tokyo, Japan), in vacuumed evaporators before examination. For observation, a Scanning Electronic Microscopic JEOL JSM-5310LV (Jeol, Tokyo, Japan) working with a voltage of 15 kV was used.
The microphotographs were carried out with a camera coupled to the microscopic.
Under all operating conditions where the details were given above, SEM photos of Ganoderma samples were taken in order to investigate the effect of temperature on physical structure of extract powders. Representative SEM micrographs, taken at magnification of 1500 are shown in Fig. 3.17 that there are distinctive differences between the molecular structures being getting smaller as a result of treatments at higher temperatures. For both batch and semi-continuous modes of hydrothermal treatment, increasing temperature gradually increased color of extracts. Optimum color and efficiency was observed at 473 K (Fig. 3.18). The destruction of cell structures by penetration of water at sub-critical conditions was clearly observed where the data might be useful elsewhere such as in modeling of the extraction.
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373 K 423 K
473 K 523 K
573 K
423 K
473 K 448 K
373 K
Figure 3.18. Effect of temperature on Ganoderma extracts for batch mode (left) and semi-continuous mode (right) hydrothermal treatments.
As in the extraction experiments, the results suggest that pressurized hot water extraction is greatly affected by extraction temperature. The representative SEM micrographs for spray dried-powdered form of extracted samples (Fig. 3.19) show that the sample components, which are mainly polysaccharides, especially β-glucans, decrease in molecular size with increasing temperatures. Moreover, the amount of polysaccharides increased with increasing temperatures.
99 Figure 3.20. Spray dried products. (sample from 373 K experiment was negligible).
423 K 448 K 473 K
Figure 3.19. SEM images of spray dried powders of G. lucidum after hydrothermal extraction at different temperatures.
Also it was also seen that the amount of product was little bit increased with high temperature (Fig. 3.20). As illustrated before, the effect of decomposition effect of temperature can be seen
from the Fig. 3.18 representing the structures of residues after
hydrothermal treatment.
373 K 423 K
448 K 473 K
100
It may be observed from Fig. 3.21 that in case of spray drying process, there are distinctive differences with amount of products, being getting smaller as a result of hydrothermal treatments at higher temperatures. After getting the Ganoderma extracts in powder form, 0.44 grams of beta glucan was detected per 100 grams of sample material referring to the relevant enzymatic methods.
Average molecular size investigation was done by MALDI-TOF MS (Voyager PerSeptive Biosystems, Tokyo, Japan) mass spectrum and it was seen that for water soluble polysaccharides showed the molecular weight distribution mostly in 500-1400 g/gmol range. Also polysaccharides up to 21 monomer units were extractable. Relevant diagram was illustrated in Fig. 3.22 representing molecular weight distribution for the best extraction condition at 473 K. there was no visible difference in molecular weight distribution among temperatures.
101
(373 K) (423 K)
(448 K) (473 K)
Figure 3.21. Powdered forms of Ganoderma lucidum after spray drying.
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Figure 3.22. MALDI-TOF mass spectrum of peaks from Ganoderma sample extracted for 130 min at 473 K.