Brazilian Journal of Chemical Engineering ENZYMATIC HYDROLYSIS AS AN ENVIRONMENTALLY FRIENDLY PROCESS COMPARED TO THERMAL HYDROLYSIS FOR INSTANT COFFEE PRODUCTION

Conventional production of instant coffee is based on solubilisation of polysaccharides present in roasted coffee. Higher process temperatures increase the solubilisation yield, but also lead to carbohydrate degradation and formation of undesirable volatile compounds. Enzymatic hydrolysis of roasted coffee is an alternative to minimize carbohydrate degradation. In this work, products obtained from thermal and enzymatic processes were compared in terms of carbohydrates and volatiles composition. Roasted coffee was extracted with water at 125 °C, and spent coffee was processed by thermal (180 °C) or enzymatic hydrolysis. Enzymatic hydrolysis experiments were carried out at 50 °C using the commercial enzyme preparations Powercell (Prozyn), Galactomannanase (HBI-Enzymes), and Ultraflo XL (Novozymes). These formulations were previously selected from eleven different commercial enzyme preparations, and their main enzymatic activities included cellulase, galactomannanase, galactanase, and β-glucanase. Enzymatic hydrolysis yield was 18% (dry basis), similar to the extraction yield at 125 °C (20%), but lower than the thermal hydrolysis yield at 180 °C (28%). Instant coffee produced by enzymatic hydrolysis had a low content of undesirable volatile compounds and 21% (w/w) of total carbohydrates. These results point to the enzymatic process as a feasible alternative for instant coffee production, with benefits including improved quality as well as reduced energy consumption.


INTRODUCTION
Coffee is a global commodity whose consumption and production are increasing every year.In the 2012/2013 crop year, worldwide production was 144.6 million sacks of 60 kg, while the international coffee trade had a turnover of about US$ 15.4 billion in 2009/10 (ICO, 2013).Coffee is consumed in many ways, including in the forms of roasted and ground coffee (RGC) and instant coffee (spray dried and freeze-dried).Although it is difficult to retain the same freshness as RGC in instant coffee beverages, their consumption has increased due to the ease of preparation (Ferdman, 2014) pare chemical composition (sugars and volatiles) of instant coffee produced from roasted arabica beans by the enzymatic process with similar products obtained by the conventional GEA-NIRO process.

Roasted Coffee
Arabica coffee beans with a medium degree of roast (10% mass loss during roasting, on a dry basis) were used in all experiments.Arabica coffee was chosen because its aromatic compounds are more sensitive to the extraction temperature employed (180 0 C) (Blank et al., 1991).The degree of roasting applied was specifically selected because it provides the release of an intense coffee aroma (Mayer et al., 2000).

Commercial Enzyme Preparations
Eleven commercial enzyme preparations with potential to hydrolyse coffee polysaccharides were evaluated: i) Cellulases: Celluclast 1.5 L (Novozymes Latin America, Brazil), Powercell, and Celumax C (Prozyn Biosolutions, São Paulo, Brazil); ii) Galactomannanases: Galactomannanase (HBI Enzymes, Japan) and Rhoapect B1-L (AB Enzymes, Germany); iii) β-glucanase: Ultraflo XL (Novozymes Latin America, Brazil); iv) Galactanases: Viscozyme L, Pectinex Ultra Clear, Pectinex Ultra SP-L, and Ultrazym AFP L (Novozymes Latin America, Brazil).Commercial pectinases were selected to search for galactanases because they usually have a side activity of these enzymes due to the presence of arabinogalactan, which is found in seeds, bulbs and leaves as a complex mixture with pectin (Luonteri et al., 2003).All the commercial preparations were characterized in terms of their main enzymatic activities according to the methods described below.

Moisture Determination
The moisture contents of all the solid raw materials and products were determined with a halogen moisture analyser (HB43-S Halogen, Mettler Toledo), which was calibrated to comply with the ISO 3726:1983method (ISO 3726,1983).

Mass Fraction of Soluble Solids
After Brix determination using a refractometer with automatic temperature compensation to 25 °C (RX-5000, Atago), the mass fractions of soluble solids present in the coffee extracts produced using the enzymatic and thermal treatments were estimated using the correlation proposed by Sivetz and Desrosier (1979).

Carbohydrate Analysis
The solubilised carbohydrates were quantified in terms of free and total sugars using HPAE-PAD (high performance anion exchange chromatography-pulsed amperometric detection), according to the methodology described in ISO 11292:1995(ISO 11292, 1995).For total sugars determination, the samples were first hydrolysed with 50 mL of HCl (1.0 M) at 98 °C for 2.5 hours.The concentration of soluble polysaccharides was estimated in terms of non-free sugars, calculated by subtracting the concentration of free sugars from the total sugars concentration.

Volatile Compound Analysis
Analysis of acetaldehyde, furfural, and 5-HMF present in a 3.0% (mass fraction) coffee extract solution was performed using HS-SPME-GC-MS (head space-solid phase microextraction-gas chromatography-mass spectrometry).The GC was fitted with an HP INNOVAX column (60 m x 320 μm x 0.25 μm).The temperature program was 40 °C for 5 min, followed by heating from 40 to 60 °C at 4 °C/min, and then from 60 to 250 °C at 8 °C/min.Helium was used as the carrier gas, at a flow rate of 1.2 mL/min.The MS detector operating conditions were an ionization energy of 70 eV, an interface temperature of 280 °C, a quadruple temperature of 150 °C, and an ion source temperature of 230 °C (Viegas and Bassoli, 2007).

Experimental Procedure
Roasted ground arabica coffee beans (RGC) were first extracted with hot water (125 °C) to produce an aroma extract (AE).The spent coffee (SC), which still contained a substantial quantity of roasted coffee polysaccharides, was used to produce soluble coffee by employing either enzymatic or thermal hydrolysis.The extracts obtained were denoted EHE and THE, respectively.A simplified flow diagram of the process is shown in Figure 2. The experimental conditions employed in each step are described below.

Thermal Extraction of Coffee Solids
Roasted ground coffee with particle sizes ranging from 0.5 to 2.0 mm was extracted in a pilot counterflow system consisting of three columns in series, with filters in the effluent streams.Hot water at 125 °C was used, with an extraction factor of 2.0 (2.0 kg of coffee extract from 1.0 kg of RGC) and an extraction cycle time of 30 min.The AE produced was immediately cooled to 25 °C and the concentrations of carbohydrates and volatiles were determined.The AE was freeze-dried, and the yield (Y) was calculated using Equations ( 1) and ( 2).The spent coffee (SC) residue remaining after thermal extraction (Figure 2) was then submitted to thermal hydrolysis or enzymatic hydrolysis, as detailed below. .
where : Y yield (%) : ss m freeze-dried coffee solids on a dry basis (g), calculated using Equation ( 2) : RGC m RGC on a dry basis (g) : AE m amount of coffee aroma extract (g) :

AE
x mass fraction of soluble solids in aroma extract.

Thermal Hydrolysis
The spent coffee (SC) was hydrolysed and extracted in a pilot counterflow system consisting of four columns with filters in the exit streams.Water at 180 °C was used, with an extraction factor of 7.0 (7.0 kg of thermally hydrolysed coffee extract from 1.0 kg of RGC) and an extraction cycle time of 30 min.The extract containing thermally hydrolysed coffee (THE) was immediately cooled to 25 °C and analysed in terms of carbohydrates and volatiles.The extract was freeze-dried, and the yield (Y) was calculated using Equations ( 1) and (3).

Enzymatic Hydrolysis
The SC residue obtained after thermal extraction (Figure 2) was dried at 60 °C to a final moisture content of 3.0%.It was then cooled to room temperature, milled in a chilled (5 °C) laboratory mill (Model A10, IKA), and sieved to obtain milled spent coffee (MSC) particles smaller than 500 μm.The enzymatic hydrolysis was carried out in a 250 mL microreactor, employing 20 g of MSC and 200 mL of citrate buffer (50 mM, pH 5.0), under mechanical agitation at 350 rpm and 50 °C.The enzymatic hydrolysis was started by adding the previously selected commercial enzyme preparations containing the highest activities in terms of cellulase, galactomannanase and β-glucanase.The amounts of enzymes added were adjusted in order to provide final mass fractions of 0.06% galactomannanase, 0.06% cellulase, and 0.06% β-glucanase (weight of commercial enzyme preparation per weight of MSC) in the microreactor.
The experiments (in triplicate) were conducted for 71 h, and the progress of the hydrolysis was followed by measuring the reducing sugars released using the DNS method (Müller, 1959).The enzymatic hydrolysis was stopped by cooling the reactor to 5 °C and the reaction mixture was centrifuged at 3000 rpm for 20 min.The supernatant containing the soluble products of the enzymatic reaction was analysed in terms of carbohydrates and volatiles, and then freeze-dried.The enzymatic hydrolysis yield was calculated using Equations ( 1) and (4). .
where : EHE m amount of enzymatic hydrolysis extract (g) :

EHE
x mass fraction of soluble solids in the enzymatic hydrolysis extract.

Statistical Analysis
The products obtained from the RGC (AE, THE, and EHE) were compared in terms of the contents of carbohydrates (arabinose, glucose, galactose, and mannose) and undesirable volatile compounds (furfural, 5-HMF, and acetaldehyde), using the Tukey's test (Box et al., 1978) with p<0.05.All calculations were performed using STATISTICA 7.1 software (Statsoft, 2005), with a confidence interval (CI) of 95%.

Selection of Commercial Enzyme Preparations
Eleven commercial enzyme preparations were evaluated to determine their cellulase, galactomannanase, β-glucanase, and galactanase activities.The results are shown in Table 1.
Powercell, Ultraflo XL, and Galactomannanase-HBI were selected to carry out the enzymatic hydrolysis step.Powercell showed the highest cellulase activities for both substrates (Table 1), Galactomannanase-HBI had the highest galactomannanase activity, and Ultraflo XL showed excellent β-glucanase activity (Table 1).Low (or zero) activities of galactanase were detected in all the enzymatic preparations.

Characterization of AE and THE
The products AE and THE (Figure 2) were analysed in terms of their contents of free (Table 2) and total sugars (Table 3).The non-free sugar contents were then obtained from these results (Table 4).The yields achieved in the thermal extraction and thermal hydrolysis steps were calculated using Equation (1) (Figure 3).The results for the product obtained by enzymatic hydrolysis (EHE) are also included in Tables 2-4 and Figure 3, but will be discussed separately.Both AE and THE showed low contents of free sugars, with overall concentrations of 1.0 and 3.7%, respectively (Table 2).In terms of total sugar composition (Table 3), THE contained about 36% of total sugars, with the highest concentration for galactose, followed by mannose and arabinose.These findings confirmed the efficiency of thermal hydrolysis for the solubilisation of polysaccharides.Leloup and Liardon (1993) also characterized total sugars in roasted arabica coffee extracted at 95 °C and obtained a value of 7.0%, which is similar to the value of 8.2% obtained here (Table 3).In the same work, a total sugars content of 20% was reported for an extract produced directly from roasted coffee at 180 °C.In the present case, the thermal hydrolysis extract was produced at the same temperature, but from spent coffee (roasted coffee that had already been submitted to thermal extraction), which therefore had a higher content of total sugars (36.2%).

Brazilian Journal of Chemical Engineering
Knowledge of the total and free sugar contents of AE and THE enabled estimation of the non-free sugar contents (Table 4), so that conclusions could be drawn about the polysaccharide contents of the extracts.Arabinose, galactose, and glucose were the main non-free sugars in AE, while galactose and mannose were found at higher concentrations in THE.THE contained about 32.8% of non-free sugar, which revealed the efficiency of thermal hydrolysis in releasing high molecular weight solubilized polysaccharides (Table 4).
Application of Tukey´s test demonstrated that the compositions of AE and THE differed in terms of free sugars, total sugars, and non-free sugars.For all the samples evaluated, galactose, mannose, arabinose, and glucose were the main sugars present, reflecting the constitutions of the three polysaccharides present in the structure of coffee (Oosterveld et al., 2003).However, the composition was also directly influenced by the extraction/hydrolysis conditions.The milder conditions employed in the thermal extraction led to a recovery that was 77% inferior in terms of soluble polysaccharides, compared to thermal hydrolysis.It is also important to highlight that polysaccharides containing mannose and galactose (Table 4) were only efficiently produced under the high temperature conditions used for thermal hydrolysis.Leloup and Liardon (1993) also reported that GMs from coffee were preferentially solubilized at higher temperatures (180 °C).This requirement for high temperature reflects the difficulty in cleaving the GM and AG structures.2.8 ± 0.5 a 2.3 ± 0.4 a 1.1 ± 0.6 b Galactose (%w/w) 2.6 ± 0.4 c 19.5 ± 2.5 a 6.7 ± 0.1 b Glucose (%w/w) 1.50 ± 0.04 a 0.6 ± 0.1 b 0.3 ± 0.2 c Mannose (%w/w) 0.8 ± 0.3 b 10.1 ± 0.8 a 1.7 ± 0.9 b non-free sugars (%w/w) 7.7 ± 1.2 b 32.8 ± 3.2 a 9.8 ± 2.8 b ND: not detected.Average ± CI (95%), n=3.Average values followed by different letters in the same line and in decreasing alphabetical order are significantly different from each other, according to Tukey's test (p<0.05).*Refers to the hydrolysate obtained after 71 h of enzymatic reaction.
The yields achieved in each step (extraction, thermal hydrolysis, and enzymatic hydrolysis) were calculated using Equation (1) and are displayed in Figure 3. Thermal hydrolysis (180 °C) and extraction produced yields of 28 and 20%, respectively.These values were in agreement with the higher carbohydrate content of THE.It is important to note that, although AE contained only 7.7% of total carbohy-   decreasing the quality of the product (Delgado et al., 2008).
The most important feature of instant coffee produced by the enzymatic route is probably the increase in quality resulting from lower concentrations of undesirable compounds, as shown in Table 4.The concentrations of acetaldehyde, furfural, and 5-HMF in EHE were even significantly lower than the values obtained for the aroma extract (AE).
These preliminary results therefore suggest that enzymatic hydrolysis is a promising alternative procedure for the production of instant coffee.The overall yield (considering thermal extraction together with enzymatic hydrolysis) was up to 39.4% (Figure 4), which is ~80% of the value achieved with the standard GEA-NIRO process.However, the enzymatic hydrolysis yield could be further improved by the identification of galactanases and other enzymes capable of breaking down the structure of arabinogalactan.In addition, the enzymatic process offers versatility: the reaction time and the enzymatic formulation can be selected in order to obtain products containing different mixtures of free sugars and polysaccharides.Nevertheless, it is fundamental to carry out a sensory analysis of the enzymatic extract to fully establish the potential of the studied alternative technology for instant coffee production.

CONCLUSIONS
Updated industrial technologies have moved towards the incorporation of Green Chemistry principles, with reduced production of environmentally harmful compounds and lower energy consumption.The instant coffee currently marketed is obtained using the GEA-NIRO process, which is characterized by maximization of the yield at the expense of high consumption of energy and degradation of solubilized polysaccharides.The findings of the present work show that spent coffee can be used to obtain instant coffee by an environmentally friendly enzymatic route, using the commercially available enzymatic preparations Powercell (Prozyn Biosolutions), galactomannase (HBI Enzymes) and Ultraflo XL (Novozymes Latin America), which exhibited the highest enzymatic activities on celullases, galactomanannases and galactanases.
Comparison of the products manufactured by the conventional thermal process and enzymatic hydrolysis showed that the yield and the composition of the solubilized material, expressed as total or specific sugar compounds, were influenced by the process conditions.For the thermal processes, temperature was the key factor governing performance: the total sugar concentration increased from 8.2% (AE produced at 125 °C) to 36.2% (THE produced at 180 °C).On the other hand, the extract obtained from the enzyme-based process at 50 °C contained 21.4% of total sugars.Compositional analysis of the products (AE, THE, and EHE) also revealed that temperature was particularly crucial for the release of polysaccharides containing galactose, and that the enzyme pool must be improved in order to provide more efficient breakdown of spent coffee polysaccharides.
Enzymatic hydrolysis of spent coffee at 50 °C with the selected enzyme preparations resulted in a yield of 18.4%.This was similar to the yield achieved using extraction at 125 °C, but was associated with a reduced content of volatiles arising from the degradation of carbohydrates, due to the lower temperature employed.A high economic value is placed on low concentrations of undesirable process by-products (such as furfural, acetaldehyde, and 5-HMF) in instant coffee, due to their impact on product quality. .

Figure 2 :
Figure 2: Flow diagram of soluble coffee production by thermal extraction, thermal hydrolysis and enzymatic hydrolysis.