β-Glucan recovery from Ganoderma lucidum by means of pressurized hot water and supercritical CO2

Accepted Manuscript Title: ␤-glucan Recovery From Ganoderma lucidum By Means Of Pressurized Hot Water And Supercritical CO2 ´ Author: Oscar Benito-Rom´an Esther Alonso Mar´ıa Jos´e Cocero Motonobu Goto PII: DOI: Reference:

S0960-3085(15)00146-7 http://dx.doi.org/doi:10.1016/j.fbp.2015.12.007 FBP 665

To appear in:

Food and Bioproducts Processing

Received date: Revised date: Accepted date:

30-6-2015 6-12-2015 16-12-2015

´ Alonso, E., Cocero, M.J., Goto, Please cite this article as: Benito-Rom´an, O., M.,rmbeta-glucan Recovery From Ganoderma lucidum By Means Of Pressurized Hot Water And Supercritical CO2 , Food and Bioproducts Processing (2015), http://dx.doi.org/10.1016/j.fbp.2015.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

 (1-3),(1-6)-D-glucans were extracted from mushroom Ganoderma lucidum  Pressurized Hot Water was used as solvent in a fixed bed unit  High temperatures favored the extraction but richness decreased dramatically

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 Mixtures PHW and supercritical CO2 improved the extractability of -glucans

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-glucan Recovery From Ganoderma lucidum By Means Of Pressurized Hot Water And Supercritical CO2

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Óscar Benito-Román1,2; Esther Alonso1*; María José Cocero1; Motonobu Goto2 Department of Chemical Engineering and Environmental Technology. Escuela de Ingenierías

Department of Chemical Engineering. Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan

corresponding author: [email protected]; +34 983 42 31 75

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Industriales (Sede Mergelina). University of Valladolid. c/ Dr. Mergelina s/n, 47011. Valladolid, Spain

ABSTRACT

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(1,3)-(1,6)--D-glucans were extracted from Ganoderma lucidum (34.2%, w/w) using pressurized hot water as solvent (P = 5 MPa) in a fixed bed laboratory scale unit. A RSM Box-

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Behnken experimental design was used to evaluate the effect of the temperature (135-175 ˚C),

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flow rate (0.7-1.3 mL/min) and solvent to biomass ratio (20-60 mL/g) on the extraction yield of

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-glucans and the content in -glucans of the final product. A dramatic effect of the temperature was observed: the higher the temperature, the higher the extraction yield; however, at temperatures above 158 ˚C the -glucan content in the final product began to decrease. It was also seen that experiments longer than 80 minutes are required to get the glucans dissolved. Finally, the effect of the flow rate on the extraction yield was not significant, indicating that external mass transport was not controlling the extraction process. The extraction conditions that maximize both extraction yield of -glucan (64.9 ± 0.8%) and led to the highest content in -glucans in the final product (61.7 ± 1.0%) are 158˚C, 1.3 mL/min and 60 mL/g. The addition of supercritical CO2 to the pressurized hot water throughout the extraction (done 2/25

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at 155˚C, 1mL/min of PHW and 40mL/g) produced a significant improvement of the extraction yield up to 72.5% in the best tested conditions; effect primarily attributed to the acidification

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of the media obtained by the supercritical CO2.

Keywords: -glucan, pressurized hot water, supercritical CO2, polysaccharides extraction,

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Ganoderma

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1. INTRODUCTION

(1,3)-(1,6)--D-glucans are non-starchy polysaccharides that exhibit good properties regarding

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to inmunomodulatory activity or the control of diseases (Askin et al., 2010). In this work, (1,3)(1,6)--D-glucans have been extracted from Ganoderma lucidum (34.2%, w/w), an extensively grown mushroom in Asia due to its reported potent bioactive properties, mainly attributed to

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the -glucans. Askin et al. (2010) reported several attempts to extract these polysaccharides

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from G. Lucidum by the use of organic solvents. The use of organic solvents presents some

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disadvantages which are related to the need of performing a purification step to remove the organic solvent, so the polysaccharides can be used in the food industry. As an alternative solvent, pressurized hot water (PHW) can be used. PHW term refers to the water in liquid state in the range 100 °C to 374 °C (critical point) by the application of pressure higher than the vapor pressure (Kronholm et al., 2007). PHW has been used by several authors to extract polysaccharides from natural matrix (Benito-Román et al., 2013), among other high added value compounds such as bioactive and nutraceuticals, essential oils, lipids, carotenoids or proteins. Density, surface tension, viscosity and diffusion coefficient of water change dramatically when changing pressure and, specially, temperature, having an effect on the mass transfer of the extraction process. PHW exhibits lower viscosity but higher diffusivity than 3/25

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water at room temperature, which favors the diffusion into the vegetal matrix and the release of compounds (Teo et al., 2010). An increase in temperature also contributes to weaken the hydrogen bonds between the carbohydrates and the solid matrix, accelerating the compound

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desorption. Moreover, high temperatures can contribute either to initiate hydrolysis processes of the already dissolved compounds or to affect the structure of the natural matrix (Kronholm

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et al., 2007), so a careful selection of the extraction conditions must be done in order to

preserve the structure of the extracted compounds. In the literature it is not possible to find

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works that deal with the extraction of -glucans from Ganoderma lucidum using the excellent properties of the pressurized hot water. Therefore it is necessary to develop a complete and

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systematic study in which the effect of the different process parameters on the extraction yield and the composition of the final extract (evaluated in terms of richness in -glucans) are

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evaluated.

The properties of the extraction media can be changed by the addition of supercritical CO2 to

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the pressurized hot water. Despite of the low solubility of CO2 in H2O and vice versa, the

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resulting mixture can be highly reactive (Springer at al., 2012): CO2 dissolved in water increases

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the availability of protons, catalyzing the hydrolysis reactions (Brunner, 2009). Thus these conditions tend to weaken the covalent bonds between the -glucan and the matrix, so the glucans are easily released and the extraction improved. It is hard to find in the literature data about the solubility of CO2 in H2O at high temperatures and pressures. Most of the studies are done at low temperatures and pressures, related to the geological CO2 sequestration and the carbonate precipitation. Springer et al. (2012) reported a solubility of 0.8% of CO2 in H2O at 150 ˚C and 5 MPa. Solubility at other different temperatures and pressures are also reported: Duan and Sun (2003) evaluated the solubility at temperatures up to 260 ˚C and pressures as high as 200 MPa and Crovetto (1991) reported the solubility of CO2 in H2O at different conditions up to the critical point. Other researchers have measured the solubility at 4/25

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temperatures up to 100 ˚C, and pressures up to 100 MPa (Diamond and Akinfiev, 2003) or 60 MPa (Spycher et al., 2003). The low solubility of the supercritical CO2 in the pressurized hot water is a challenge, which will

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make difficult the performance of the extraction using that mixture of solvents. It is important to evaluate the flow pattern of the mixture pressurized hot water-supercritical CO2 in the

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reactor. For instance, if a pulsed flow is detected, the changes in the extraction yield can be attributed to the turbulence generated in the reactor, contrary to the acidification effect

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primarily considered.

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The purpose of the present work was to study the effect of operational variables in the extraction of -glucans from Ganoderma lucidum using pressurized hot water as solvent. The

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influence of temperature, extraction time and the flow rate on extraction yield and content in -glucans of the final product was evaluated. In a second step of the work, CO2 was added to

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and the richness was evaluated.

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the PHW at different conditions, and the effect of this addition on both the extraction yield

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2. MATERIALS AND METHODS

2.1. Raw Material

Finely milled Ganoderma lucidum mushroom (CMT-Vibrating Sample Mill TI-100, CMT Co Ltd, Tokyo, Japan) was used in this work. Ganoderma lucidum contained 34.2 % in -glucan (moisture 11.0 %) and average particle size after milling was 26.5 ± 1.6 m (Malvern Mastersizer 2000, Malvern Ltd).

2.2. Experimental set-up 5/25

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Extraction was performed in a 10 mL fixed bed extractor, placed in an oven in order to keep the extraction at the desired temperature. In each experiment, 2 grams of the mushroom were loaded in the reactor. A total of 3 g of glass beads were incorporated in the extractor (half of

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them in the bottom and the rest on the top, in order to prevent the compression of the biomass over the sinter plate). Water was pumped by means of a HPLC pump at a constant

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flow rate and the pressure in the reactor (5MPa in all the experiments) was controlled by means of a back pressure regulator. The liquid extract obtained was cooled down in an ice

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bath in order to prevent degradation after the extraction. After the operation time, pump was stopped and the reactor was suddenly depressurized and cooled down. The solid residue was

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weighted and dried. The liquid extracts obtained throughout the experiments were kept at 4 °C and subsequently analyzed to determine the content in both -glucans and total solid

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material. This allowed to determine the total amount of biomass dissolved during the extraction and to calculate the content in -glucans of the solid product (a measure of the

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richness in -glucans of the dried extract).

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When the extraction was done using a mixture of PHW and supercritical CO2, CO2 was pumped

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by means of a syringe pump model 260D (Teledyne ISCO, 266 mL). PHW and CO2 were mixed at the entrance of the extractor. Pressure used in these experiments was in the range 510MPa. A scheme of the experimental set-up used in this work is shown in Figure 1.

2.3. Chemical analysis

-glucan content in the Ganoderma lucidum (expressed in grams of -glucan per 100g of mushroom) and in the liquid after the extraction was determined by means of the assay kit provided by Megazyme Ltd. (Ireland) “Mushroom and Yeast -glucan”. This assay kit allows to know the amount of -glucan in the liquid extract, expressed in grams of -glucan per 100g of 6/25

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liquid extract. Then, once it is known the grams of -glucans extracted under a given conditions, the extraction yield was calculated according to the equation 1:

(1)

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g β-glucan content in liquid extract 100g · Liquid Extract (g) Extraction Yield (%)= x 100 g β-glucan content in G. lucidum · G. lucidum (g) 100g

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The effect of the process parameters on the content in -glucans of the extract was also

evaluated according to equation (2). This equation provides the richness in -glucans that has

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the final extract, expressed in dry basis. This value relates the amount of -glucans extracted with the total co-extracted compounds, indicating how effective is the extraction process to

β-glucan extracted (g)

g Liquid extract (g)·Solid content g

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Extract Content in β-glucan (%)=

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concentrate the extract in -glucans.

x 100

(2)

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The solid content of the liquid extract was determined by gravimetry: a known mass of liquid

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extract was set in an oven at 105 ˚C for 24h. After that the solid residue was weighted and the

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% of solid material in the liquid extract calculated.

2.4. Experimental work

The experimental work was split in two different stages: initially a RSM Box-Behnken experimental design was used to evaluate the effect of the process parameters on the extraction yield and the content in -glucans of the extract (the richness in -glucans): X1, temperature (135-175 ˚C); X2, flow rate (0.7-1.3 mL/min) and X3, solvent to solid ratio (20-60 mL water/g of Ganoderma lucidum). The solvent to solid ratio indicates the total amount of water used in the experiment to carry out the extraction. It must be noted that the extraction time can be calculated by dividing the total amount of solvent used in the experiment by the flow rate. Therefore the extraction time is not a process parameter but a consequence of 7/25

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other two operational parameters. In this stage of the experimental work water was used as solvent. A statistical analysis of the experimental results was done using the statistical software Statgraphics Centurion XVI, defining a confidence level of 95%.

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In the second stage of the experimental work, the central point of the experimental design (155 ˚C, 1 mL/min and a total amount of water of 80 mL) was used to evaluate the effect of the

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addition of CO2 on the extraction yield of -glucans and richness of the extract in β-glucans. CO2 flow rate was set at 1 mL/min and three different pressures were tested in the range 5

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MPa (below the critical point) to 10 MPa.

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3. RESULTS AND DISCUSSION

3.1 Pressurized hot water extraction

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The experimental plan, as well as the results regarding to the -glucan extraction yield, the

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content in -glucan of the final extract (richness in -glucans, expressed in dry basis) and the final pH of the liquid extract, are shown in table 1. Extraction yield was in the range 8.9-79.3%

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(experiments 5 and 8) meanwhile the richness was in the range 24.0-62.2% (experiments 5 and 12). No significant differences were observed in the final pH of the liquid extract, despite of the different experimental conditions tested. A deeper discussion about the effect of the process variables will be done in the following paragraphs. 3.1.1.

Effect of the extraction conditions on the -glucan extraction yield

The extraction process of the -glucans from natural matrixes is complex, since they are strongly linked to the matrix. Hence, in order to recover high amounts of the -glucans, it is necessary to break those bonds: first hydrolysis must happen, and then the -glucan is solubilized. Significant differences were observed in the obtained extraction yield. Figure 2 8/25

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shows the main effects plot for the extraction yield: it includes the effect that a change in one of the process parameters has on the extraction yield. It can be clearly seen that the extraction yield is not affected by the flow rate, but mainly affected by the extraction temperature and

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the solvent to solid ratio (which at the end represents the extraction time for a given flow rate). The effect that both parameters have on the extraction yield has the same trend: the

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higher the temperature the higher the extraction yield and the higher the amount of solvent

passed through the Ganoderma lucidum bed, the higher the extraction yield. However, the use

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of high quantities of solvent can be a problem if a further purification/isolation step is considered, since more solvent will have to be removed. On the other hand, the effect of the

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flow rate on the extraction yield was more limited compared to the other variables.

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Results of the analysis of variance are presented in Table 2, they can be used to discuss the interaction between factors. From Table 2 it can be inferred that temperature and the solvent

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to solid ratio (which determines the amount of solvent used in the experiment) have a

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statistically significant effect on the extraction yield; this means that temperature and amount

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of solvent are controlling the extraction of -glucans. However, no interaction between the studied factors was observed.

High temperatures have a double effect: help to weaken the hydrogen bonds between glucans and the matrix where they are trapped, and help to increase the solubility of the glucans. It is known that the transport properties of water change dramatically when increasing the temperature: a decrease in surface tension and viscosity happens; so water can penetrate easily in the matrix, break the bonds between the -glucan and other molecules that form part of the structure and hence -glucans can be easily dissolved and extracted. A linear increase of the -glucans recovery with temperature has been found in the extraction from Ganoderma lucidum, contrary to the results obtained in the extraction of (1,3),(1,4)--D9/25

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glucans from barley in a previous work (Benito-Román et al., 2013). -glucan recovery from barley increased with temperature up to 155-160 ˚C (Benito-Román et al., 2013) presenting a decrease in the extraction yield beyond this temperature. In the case of barley, -glucans

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suffered severe degradation (by the loss of molecular weight and the presence of sugar degradation products). However -glucans from Ganoderma lucidum present a stronger

Effect of the extraction conditions on the extract richness in -glucan

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3.1.2.

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structure, so higher temperatures can be used to extract them.

A successful extraction process must provide the highest extraction yield of the target

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compound and lead to the highest richness of the target compound in the final product in order to facilitate the downstream purification step that removes the co-extracted

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compounds. This will indicate that the extraction process has been useful to preferably extract the target molecule and not others. Ganoderma lucidum, as well as other natural matrices, is

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very complex: many different compounds such as proteins, 12-15%; total carbohydrates 75-

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85%, among them -glucans and cellulose; fats 3%; ash <2% according to Hung and Nhi (2012) are present and depending on the extraction conditions can be co-extracted together with the The main effects diagram (Figure 3) shows the effect that process

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target compound.

parameters have on the content in -glucans in the final extract (expressed in dry basis).

Table 3 shows the ANOVA study for the effect that the different process parameters have on the content in -glucans of the final extract. Both the temperature and the amount of solvent used in the experiment have a statistically significant effect on it, and there was a strong interaction between those two parameters and between the flow and the amount of solvent used (the solvent to solid ratio). The effect that the temperature has on the compositions of the extract is clear; the higher the temperature the higher the richness of the extract in -glucan, but up to a maximum reached 10/25

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at 158-160 ˚C; beyond this temperature the extract is not only not further enriched in glucans but the richness begins to decline. This fact indicates that other compounds present in the structure of the mushroom begin to be more preferably extracted. The effect that the

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amount of solvent has on the richness in -glucans of the final extract is also clear: the higher the quantity of solvent the higher the enrichment. In order to release the-glucans, the water

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has to penetrate into the Ganoderma lucidum structure and weaken the hydrogen bonds between -glucans and the structure.

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With the flow rate and the solvent to solid ratio (total amount of solvent), it is possible to

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calculate the extraction time. As it has been seen, high flow rates and low solvent to solid ratios (which involves the shortest experiments) led to low enriched extracts in -glucans;

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being this enrichment lower when temperature is decreased: only those compounds that are not strongly linked to the matrix are extracted; under these conditions if the extraction

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temperature is increased, the enrichment of β-glucans is increased as well. This phenomenon

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can be explained since the temperature improves the extraction of -glucans. However, if working at low flow rates the amount of solvent used in the extraction is increased, the

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enrichment increases dramatically as the experiment is longer and more water is used (higher gradient concentration throughout the extraction experiment). Nevertheless, at 175 ˚C it is observed a decrease in the concentration of -glucans in the final extract, indicating the presence of other extraction compounds. When high amounts of solvent are used, the effect of the flow rate becomes less important, being the extraction slightly less selective to the glucan at the highest flow rate.

All these phenomena described in the previous paragraphs are shown in Figure 4, where the extract richness in -glucans of the final extract (expressed in %, w/w, dry basis) as a function of the extraction time is shown at different temperatures.

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From the experimental results presented in Table 1 and the statistical analysis presented in table 3, it can be seen that the flow rate shows a much more attenuated effect on the -glucan enrichment process (its effect is not statistically significant): at a given temperature and

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amount of solvent, an increase of the flow rate slightly decreases the richness of the -glucan in the extract.

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All in all, a careful selection of the extraction conditions has to be done, considering that the concentration of -glucans in the final extract is mainly affected by the temperature and the

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amount of solvent used in the experiment. Figure 5 shows the effect that temperature and

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solvent to solid ratio have on the concentration of -glucans in the final extract using PHW as solvent and Ganoderma lucidum as raw material. It can be seen clearly that there is a range of

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experimental conditions that maximize the concentration: temperature in the range 150-160˚C and solvent to solid ratios above 40 mL/g (leading to extraction experiments that demand at

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least 80 mL of solvent and at least 80 minutes). Higher temperatures decrease the concentration in the final extract, as other compounds begin to be extracted together with the

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-glucans.

3.1.3.



-glucan extraction optimization

From the separate study of the effect that the process parameters have on the extraction yield and the concentration of -glucans in the final extract when the mushroom Ganoderma lucidum is used as raw material, it can be concluded that the -glucan extraction process is long, taking more than 80 minutes to get high extraction yields and high concentrations of the target compound in the final extract. Large amounts of water are required to extract the glucans in order to swell the cells and long times are also required to weaken the hydrogen bonds. The process might be accelerated by increasing the temperature, the -glucans might 12/25

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not be damaged but the selectivity of the extraction process would be reduced, as other compounds would be extracted together with the -glucans. So it is necessary a compromise between the amount of -glucan extracted (extraction yield)

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and the richness of -glucans in the final extract: higher percentages of the target compound in the final extract will reduce significantly the subsequent purification step. Based on these

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requirements, it was calculated the optimal conditions (temperature, flow rate and solvent to solid ratio) that maximized extraction yield and the enrichment. For that purpose the data

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obtained in the Box–Behnken experimental design (described in Table 1) for each response

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variable were converted into a second-order polynomial equation with three independent variables, using the statistical software Statgraphics Centurion XVI. The multiple coefficient of

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correlation (R2) obtained for each linear regression model (extraction yield and richness) is 0.9783 and 0.9886, respectively. This value indicates a close agreement between experimental and predicted values; so it was used to calculate the optimum conditions, shown in table 4. A

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cross-check experiment was performed at the calculated optimal conditions, and as can be

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seen in table 4, there is a close agreement between the predicted and the observed results.

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To maximize both extraction yield and the richness in -glucans of the final extract, the key parameter is the temperature. It has to be in the range 155-160˚C. 3.2 Pressurized hot water and supercritical CO2 extraction

As it has been shown in previous experiments described in section 3.1, the use of pressurized hot water allows getting high extraction yields of -glucans from Ganoderma lucidum. PHW has different chemical properties compared to water at room temperature. Among them, there is a dramatic change in the ionic product, increasing from 10-14 at room temperature to 10-11.2 at 155 °C. This leads to an increase in the concentration of the hydronium ions which promote the hydrolysis reactions (Plaza and Turner, 2015). This helps to increase the extraction rate of -glucans. 13/25

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In order to improve the extraction of -glucans from Ganoderma lucidum, CO2 at different pressures (50-85-100 bar) was added to the pressurized hot water at a constant flow rate of 1 mL/min. The purpose of this addition was to produce a further acidification of the extraction

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media in order to increase the extraction rate of -glucans. Duan and Sun, (2003) reported the calculated solubility of CO2 in water under different conditions; at 155 °C and 50, 85 and 100

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bar these values are 0.413, 0.664 and 0.771 mol/L, respectively. These results would lead to pH values below 4, clearly lower than those obtained when only water is used as a solvent.

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All the experiments were done in the central point of the experimental design: 155 ˚C, 1

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mL/min of PHW and a solvent to solid ratio of 40 mL/g. When no CO2 had been added, the extraction yield under those conditions was 54.0 ± 2.0% and the content in -glucans of the

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final extract (expressed in dry basis) 55.0 ± 1.0%. Results are shown in Figure 6, where the effect of the addition of CO2 at different pressures on the extraction yield and on the

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concentration in -glucans of the final extract are presented.

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When CO2 at 5 MPa was added an increase of the extraction yield was seen up to 58.6%, from

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the 54.0% when no CO2 was added. The addition of CO2 increased the extraction yield as a consequence of the pH decrease that suffered the extraction solvent. When pressure was increased and CO2 reached the supercritical state, bigger increases in the extraction yield and the richness in -glucans were observed. Above the critical pressure of CO2, extraction yield was dramatically improved (above 70%), and richness in -glucans was also increased up to 60%, and both of them remained almost constant at a higher pressure. As it has been described, the increase in pressure only produced a slight increase in the solubility of CO2 in water promoting a very limited effect on the pH diminution. However, when CO2 reaches the supercritical state, its properties suffer dramatic changes: for instance, at 155 °C and 5 MPa, its density is 66 kg/m3 whereas at 155 °C and 8.5 MPa it increases up to 118 kg/m3 and further 14/25

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increases of pressure up to 10 MPa increases the density up to 144 kg/m3. Therefore, the increase in the extraction yield when pressure is above the critical value can be attributed to the changes in the flow pattern in the fixed bed associated to the changes in the CO2 density.

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Flow pattern was calculated and it was seen that, under the experimental conditions, it was a slug flow, with bubbles of supercritical CO2 (Hewitt and Roberts, 1969). The formation of

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bubbles improves the extractability of -glucans, since these bubbles of higher density help to expand the matrix (Ganoderma lucidum). Moreover, physical and morphological properties of

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the polysaccharides such as -glucans, can be modified by the presence of supercritical CO2, as

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it happens in other polymers (Erdogan, 2009).

The addition of supercritical CO2 to the PHW improved the extraction yield of -glucans, up to

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a maximum of 72.5% when operating at 8.5 MPa, and the content in -glucans of the final extract of the extraction was only improved up to 58%. This result showed that the addition of

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supercritical CO2 to the PHW tends to improve the extraction of -glucans, but also that of

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other compounds present in the matrix and therefore the improvement in richness is negligible. When the extraction was done using only PHW as solvent (1 mL/min, 155 °C, 8.5

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MPa and 40mL/g) the extraction yield and the richness values were 58.3 ± 1.2% and 56.7 ± 1.7%, respectively. Table 5 shows a comparison between the extraction results when the extraction was done with and without CO2. It can be concluded that the addition of CO2 to the extraction medium increases the extraction yield and introduce a slight increase in the concentration of -glucans in the final extract.

4. CONCLUSIONS (1,3)-(1,6)--D-glucans were successfully extracted from the mushroom Ganoderma lucidum by means of pressurized hot water. The effect of different process parameters was studied, 15/25

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and concluded that the extraction temperature played a dramatic role on the extraction performance: the higher the temperature the higher the extraction yield; however, high temperatures led to a decrease of the content in -glucans of the final extract (expressed in

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dry basis). At low temperatures (135 °C) only those -glucans easily accessible for the solvent are extracted and solubility is controlling the process. Higher temperatures produce an

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increase of the extraction rate of -glucans. To maximize both extraction yield and concentration of -glucans in the final extract, temperature has to be in the range 155-160 ˚C

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with an extraction time of around 92 min. Under these conditions the extraction yield is

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64.9±0.8% and richness in -glucan of the extract is 61.7±1.0.

Finally, the addition of CO2 to the pressurized hot water enhanced the extraction yield and

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increased slightly the richness in -glucans of the extract. The mechanism that explains this improvement can be explained on one hand by the increase of proton availability in the

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extraction media caused by the CO2 dissolved in water, and on the other hand, by the

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expansion of the matrix and the change in flow pattern by bubble formation when pressure is above the supercritical value. All in all the use of pressurized hot water and supercritical CO2 as

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an extraction solvent is a smart way to induce the acidification of the medium as, once the solvent after the extraction is depressurized, the CO2 is released (passing from supercritical to gas state), not staying in the liquid extract. If a mineral or organic acid is used they remain in the liquid after the extraction, being necessary a purification step in order to remove them.

5. ACKNOWLEDGEMENTS The authors want to thank the University of Valladolid fellowship research program FPI-UVa, for the financial support for the stay at the Nagoya University.

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6. REFERENCES Askin R., Sasaki M., Goto M., 2010. Recovery of water soluble compounds from Ganoderma

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lucidum by hydrothermal treatment. Food Bioprod. Process. 88, 291-297. Benito-Román Ó., Alonso E., Cocero M.J., 2013. Pressurized hot water extraction of β-glucans

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from waxy barley. J. Supercrit. Fluid. 73, 120-125.

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Brunner G., 2009. Near critical and supercritical water. Part I. Hydrolytic and hydrothemal processes. J. Supercrit. Fluids 47, 373-381.

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Crovetto R., 1991. Evaluation of Solubility Data of the System CO2-H2O from 273K to the Critical Point of Water. J. Phys. Chem. Ref. Data 20(3), 575-589.

M

Diamond L. W., Akinfiev N. N., 2003. Solubility of CO2 in water from 1.5 to 100˚C and from 0.1

208, 265–290.

d

to 100 MPa: evaluation of literature data and thermodynamic modeling, Fluid Phase Equilibr.

te

Duan Z., Sun R., 2003. An improved model calculating CO2 solubility in pure water and aqueous

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NaCl solutions from 273 to 533 K and from 0 to 2000 bar. Chem. Geol. 193, 257– 271 Erdogan K., 2009. Polymer miscibility, phase separation, morphological modifications and polymorphic transformations in dense fluids. J. Supercrit. Fluid. 47, 466-483. Hewitt G.F., Roberts D.N., 1969. Studies of Two-Phase Flow Patterns by Simultaneous X-ray and Flash Photography, AERE-M-2159, Technical Report, Atomic Energy Research Establishment.

Ho C.H.L., Cacace J.E., Mazza G., 2007. Extraction of lignans proteins and carbohydrates from flaxseed meal with pressurized low polarity water. LWT-Food Sci. Technol. 40, 1637-1647. Hung P. V., Nhi N.N.Y., 2012. Nutritional composition and antioxidant capacity of several edible mushrooms grown in the Southern Vietnam. Int. Food Res. J. 19(2), 611-615. 17/25

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Kronholm J., Hartonen K., Riekkola M.L., 2007. Analytical extraction with water at elevated temperatures and pressures. Trends Anal. Chem. 26(5), 396-411. Plaza M., Turner C., 2015. Pressurized hot water extraction of bioactives. Trends Anal. Chem.

ip t

71, 39-54. Springer R.D., Wang Z., Anderko A., Wang P., Felm A.R., 2012. A thermodynamic model for

cr

predicting mineral reactivity in supercritical carbon dioxide: I. Phase behavior of carbon

us

dioxide–water–chloride salt systems across the H2O-rich to the CO2-rich regions. Chem. Geol. 322-323, 151-171.

an

Spycher N., Pruess K., Ennis-King J.,2003. CO2-H2O mixtures in the geological sequestration of CO2. I. Assessment and calculation of mutual solubilities from 12 to 100°C and up to 600 bar.

M

Geochim. Cosmochim. Ac. 67(16), 3015–3031.

Teo C.C., Tan S.N., Hong Yong J.W., Hew C.S., Ong E.S., 2010. Pressurized hot water extraction

Ac ce p

te

d

(PHWE). Review, J. Chromatogr. A 1217, 2484-2494.

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Page 18 of 31

ip t cr

Experimental Conditions X2 X3 X3/X2* Ext. Time (min) F (mL/min) s:s (mL/g) 0.7 40 114

1

X1 T (°C) 135

2

175

0.7

40

3

135

1.3

40

175

1.3

40

135

1.0

20

6

175

1.0

20

7

135

1.0

60

8

175

1.0

60

Experimental Results Richness** Solid Content*** (%) (%) 41.3 0.355

pH 4.24

114

73.2

49.9

1.255

4.00

62

12.1

40.7

0.254

3.89

62

71.2

49.8

1.223

3.80

40

8.9

24.0

0.635

4.05

40

55.3

44.5

2.216

3.76

120

24.3

49.3

0.281

4.22

120

79.3

50.1

0.903

3.97

ed

4 5

Yield (%) 17.2

M an

Run

us

Table 1. Experimental conditions, extraction yield, richness, solid content and pH of the 15 experiments that form the Box–Behnken experimental design.

155

0.7

20

57

36.6

44.3

1.413

3.82

155

1.3

20

31

24.6

41.5

1.014

3.87

11

155

0.7

60

171

68.9

53.8

0.731

4.06

12

155

1.3

60

92

63.7

62.2

0.584

3.81

13

155

1.0

40

80

53.5

53.4

0.685

3.82

14

155

1.0

40

80

52.3

54.7

0.654

3.81

15

155

1.0

40

80

56.2

56.9

0.676

3.81

ce pt

9 10

* Extraction time calculated as a function of the flow rate and the, solvent to solid ratio used in each experiment

Ac

** Richness: refers to the -glucan content in the final extract, expressed in dry basis *** Solid content in the liquid extract

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Page 19 of 31

Table 2. ANOVA table for the extraction yield results Source A: Temperature

DF

Mean Square

F-Value

p-Value

5867.110

1

5867.110

169.46

0

1

73.694

2.13

0.2044

1532.990

1

1532.990

44.28

0.0012

AA

269.636

1

269.636

7.79

0.0384

AB

2.4016

1

2.402

0.07

0.8028

AC

18.399

1

18.399

0.53

BB

14.902

1

14.904

0.43

BC

11.584

1

11.584

0.33

CC

44.823

1

44.823

Error total

173.113

5

34.623

Total (corr.)

7983.54

14

ip t

73.694

C: Solvent to solid ratio

0.4987

0.5408

cr

0.5881

0.3068

us

1.29

Ac ce p

te

d

M

an

B: Flow

SS

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Page 20 of 31

Table 3. ANOVA table for the final extract richness in -glucan results Source A: Temperature

DF

Mean Square

F-Value

p-Value

1

189.467

72.76

0.0004

1

3.121

1.20

0.3235

465.645

1

465.645

178.8

0.0000

AA

301.506

1

301.506

115.77

0.0001

AB

0.0621

1

0.062

0.02

0.8833

AC

97.540

1

97.540

37.45

BB

1.052

1

1.052

0.40

BC

31.776

1

31.776

12.20

CC

59.059

1

59.059

Error total

13.021

5

2.605

Total (corr.)

1143.290

14

ip t

3.121

C: Solvent to solid ratio

0.0017 0.5530

cr

0.0174 0.0050

us

22.68

Ac ce p

te

d

M

an

B: Flow

SS 189.476

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Page 21 of 31

Table 4. Predicted and experimental results obtained under the calculated optimum conditions

1.3

Richness (%)

Predicted

Observed

Predicted

Observed

65.4

64.9±0.8

61.3

61.7±1.0

60

ip t

158

Extraction Yield (%)

Ac ce p

te

d

M

an

us

cr

Maximized Response Extraction Yield & Richness

Process Variables Solvent to T F solid ratio (˚C) (mL/min) (mL/g)

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Page 22 of 31

Table 5. Extraction results when CO2 was and was not added to the PHW for the extraction of -glucans EXPERIMENT ID 2 155 ˚C – 8.5 MPa PHW (1 mL/min) PHW (1 mL/min) + CO2 (1 mL/min) 58.3 ± 1.2 72.5 ± 3.5 56.7 ± 1.7 58.0 ± 2.6 3.81 ± 0.01 3.88 ± 0.01

Ac ce p

te

d

M

an

us

cr

Conditions Solvent Extraction yield (%) Concentration (%) Final pH

ip t

1

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Page 23 of 31



Figure 1. Experimental set-up used in this work

 Figure 2. Main effects diagram for the extraction yield of -glucans from Ganoderma lucidum. Effect of temperature (), flow rate () and solvent to solid ratio () on extraction yield

 Figure 3. Main effects diagram for the concentration of -glucans extracted from Ganoderma

ip t

lucidum in the liquid extract (expressed in % w/w, dry basis). Effect of temperature (), flow rate () and solvent to solid ratio () on extraction yield

 Figure 4. Concentration of -glucans in the extract (expressed in % w/w, dry basis) as a function

cr

of the extraction time at different temperatures

 Figure 5. Surface diagram for the selectivity of the -glucans extraction from Ganoderma

Figure 6. Effect of the operation pressure on the extraction yield () and the concentration of -glucans in the liquid extract expressed in %, w/w, dry basis (), at 155 °C, using 1 mL/min of PHW and 1 mL/min of CO2 at different pressures

Ac ce p

te

d

M

an



us

lucidum in the liquid extract (expressed in % w/w, dry basis). The effect of the extraction temperature and solvent to solid ratio on the extraction yield at constant flow rate (1mL/min)

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Page 24 of 31

us

cr

ip t

Ganoderma lucidum

Enriched extract in (1,3)(1,6)--D-glucan

an

water

Ac ce p

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d

M

CO2

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Page 25 of 31

Figure 1

TI

PI

102

101

TI

103 E-101

ip t

V-101

cr

TI

P-101

101

Sampling

us

water

P-102

Ac

ce pt

ed

M

an

CO2

Page 26 of 31

Figure 2

80,0 70,0

50,0 40,0 30,0 20,0 10,0 0,0 155

175

1

1,3

F (mL/min)

20

40

60

s:s ratio (mL/g)

Ac

ce pt

ed

M

an

us

T (ºC)

0,7

cr

135

ip t

Extraction Yield (%)

60,0

Page 27 of 31

Figure 3

60,0 55,0

b- glucan (%)

50,0 45,0 40,0 35,0

ip t

30,0

20,0 135

155

175

20

40

60

s:s ratio (mL/g)

Ac

ce pt

ed

M

an

us

T (ºC)

0,7 1 1,3 F (mL/min)

cr

25,0

Page 28 of 31

Figure 4

60,0

40,0 30,0

ip t

b-glucan (%)

50,0

20,0

135ºC 155ºC

cr

10,0

175ºC

0

50

100

150

200

Ac

ce pt

ed

M

an

time (min)

us

0,0

Page 29 of 31

55

48

175

155

27 20

145

s:s ratio (mL/g G.lucidum)

us

T (ºC)

34

cr

41

165

57,0-62,0 52,0-57,0 47,0-52,0 42,0-47,0 37,0-42,0 32,0-37,0 27,0-32,0 22,0-27,0

ip t

b-glucan (%)

Figure 5

Ac

ce pt

ed

M

an

135

Page 30 of 31

70,0

70,0

60,0

60,0

50,0

50,0

40,0

40,0

30,0

30,0

20,0

20,0

10,0

10,0

0,0

0,0

ip t

80,0

b-glucan concentration (%)

80,0

4

5

6

7

8

9

10

Ac

ce pt

ed

M

an

us

Pressure (MPa)

cr

Extraction Yield (%)

Figure 6

Page 31 of 31