A Laboratory Exercise To Understand the Importance of Enzyme

In the Laboratory

A Laboratory Exercise To Understand the Importance of Enzyme Technology in the Fruit-Processing Industry: Viscosity Decrease and Phenols Release from Apple Mash Manuel Pinelo, Michael K. Nielsen, and Anne S. Meyer* Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Søltofts Plads, Building 229, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark *[email protected]

Students in technology-based laboratory courses can benefit from simple experiments that illustrate the advantages of using enzymes in industrial processes. In industrial apple juice processing, enzymes are used in the fruit maceration step to decrease viscosity primarily by pectin hydrolysis. The enzymes used are commercial, microbially produced enzyme preparations, typically pectinases derived from Aspergillus spp. These enzyme preparations are mixtures of several different enzymes that contain the main pectinases, pectin methyl esterase, endopolygalacturonase, and pectin lyase, and also contain various other enzymes such as galactanases, arabanases, and cellulases.1 These enzymes attack the plant cell-wall polysaccharides present in fruit cell walls and promote the degradation of polysaccharides via specific enzymatic catalysis. The enzymatic action lowers the fruit mash viscosity to ease the pressing of the mash, increase juice yields, and at the same time enhance the release of antioxidant phenols from the raw materials into the juice (1). The present exercise has been part of our laboratory course in advanced enzyme technology2 for the last four years and has a number of important advantages: First, the raw material (apple) and the enzyme preparations are inexpensive and commercially available. Second, the students perform a practical enzymatic maceration that results in rapid viscosity lowering of the fruit mash, which is measured instrumentally but can also be seen directly by the naked eye. Third, the students assess the extraction of antioxidant phenols into the juice. It is our experience that the prospects of assessing putative health promoting compounds such as antioxidants make the exercise particularly interesting for the students. Fourth, the exercise provides good opportunities to discuss the challenges of relevant quantitative assessments of the enzyme activity of pectinases and offers simple, practical solutions to analysis of complex data. Finally, the whole exercise can be accomplished within a 4-h laboratory exercise session; the data analysis and discussions of the theory are then done in a subsequent teaching session. The purpose of the exercise is to demonstrate that certain enzyme preparations catalyze the degradation of polysaccharides, notably the pectin present in the primary cell walls and as the intercellular putty (middle lamella) that connects the apple pulp cells. This enzyme-catalyzed degradation makes the juice pressing easier and results in an elevated release of phenols from the raw apple mash into the juice. During the exercise, the students (i) perform an accelerated enzymatic maceration, (ii) understand and assess the efficiency of certain different types of enzyme

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preparations in catalyzing the degradation of the apple material (based on viscosity measurements) that leads to release of polyphenols (based on a specific analytical method), and (iii) evaluate the effects of different types of enzymes on the apple mash viscosity and on the release of the phenols. Background Many types of phenols are naturally found in plants and hence in edible plant-based products, including apples, and they may have a role in the correlation between high fruit and vegetable consumption and reduced risk of certain cancers and cardiovascular disease (2, 3). Phenols, which contain a six-membered ring with an -OH group, are classified into different categories depending on the carbon side groups and the number and position of their hydroxyl groups (Figure 1). Apple phenols include (a) benzoic acids such as gallic acid, (b) hydroxycinnamic acids such as caffeic and chlorogenic acids, (c) flavonols such as quercetin, (d) flavan-3-ols also known as catechins (e.g., epicatechin and catechin), and (e) dihydrochalcones such as phloridzin (1) (Figure 1). The cell walls in fruits (dicot plants) are composed of three structurally independent, interacting parts: a fundamental cellulosexyloglucan framework that is embedded in a matrix of pectin polysaccharides and intertwined with structural proteins (4). The phenols are bound in the plant cell wall in different ways, but cellulases, pectinases, proteases, and other enzymes able to catalyze the cleavage of bonds in plant cell-wall polysaccharides can be employed to decompose the cell-wall structure and improve the release of phenols entangled in the plant material matrix (5). Experiment Preparation of the Sample Each student (or group of students) washed, destemmed, and cut three fresh golden delicious apples into four pieces. They then used a meat grinder (e.g., Jupiter type 863, Germany) to obtain the apple mash (apples were not peeled). Alternatively, a blender or another kind of grinder could be used. A 50:50 mixture of potassium metabisulfite and ascorbic acid (1 g each compound/kg of apple) was immediately added to the apple mash to prevent browning. Enzyme Maceration and Viscosity Measurement The enzyme preparation, added at a dosage of 0.01% enzyme/substrate (mass/wet mass), was added to 30 g of apple

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r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 88 No. 4 April 2011 10.1021/ed100240s Published on Web 12/20/2010

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In the Laboratory

mash. First, a sample without enzyme was prepared (control). Subsequently, the following enzyme treatments and a combination were prepared: (i) pectinase, (ii) cellulase, and (iii) pectinase and cellulase (Table 1).3 For each enzyme treatment, the maceration was performed in duplicate or triplicate. The maceration was performed in a rapid visco analyzer (RVA) (Newport, U.K.) at 50 °C (optimal temperature for the enzymatic actions), maintaining the agitation at 900 rpm during the first 10 s of maceration to ensure efficient mixing, followed by agitation at 160 rpm for 5 min. The advantage of the RVA is that the viscosity is monitored in real-time and can be viewed on a computer, but the viscosity can also be assessed by other means, for example, by use of a classical Brookfield viscometer, a rheometer, or any other viscosity measurement. If the particular viscometer does not allow real-time measurements, differences in viscosity can just be evaluated by timed measurements of the viscosity before and after the enzymatic maceration. The viscosity data were employed to define the efficiency of the enzyme preparations on the cell-wall decomposition. The enzymes were then inactivated by subjecting the mash to a 90 °C heat treatment for 1 min; the apple mash was then pressed and the resulting juice was obtained by centrifugation at ∼15,000g for 10 min. It is not necessary to measure the volume or assess the clarity of the juice obtained. Determination of the Released Phenols The quantity of released phenols was assessed by the classic Folin-Ciocalteu assay (6). A small volume, 1 mL, of 10-fold diluted Folin-Ciocalteu reagent was added to a 0.2 mL of 1:10 diluted juice sample. After 1 min, 0.8 mL of 7.5% (w/v) of

Figure 1. Principal structures of phenols found in green and yellow apples; mainly in the apple skins.

Na2CO3 solution was added and the mixture was shaken. After 30 min, the optical density was measured at 765 nm. The phenol concentration was expressed as gallic acid equivalents (GAE mg/L) using gallic acid (Sigma-Aldrich, St. Louis, MO) as a standard (6). Hazards Folin-Ciocalteu reagent is toxic and is harmful if inhaled, absorbed through skin, or swallowed. Students should wear gloves, avoid direct contact with the reagent and work under a fume hood. The liquids must be carefully discarded in special containers after the analyses have been done. The enzymes and potassium metabisulfite may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. Results Viscosity Variation during Enzymatic Maceration During the maceration of apple mash without enzymes, a rapid decrease in viscosity was observed during the first minute, which was followed by a progressive, slower decrease over the remaining 5 min (Figure 2). The use of enzymes, both individually and in combination, promoted a continuous decrease in the viscosity of the apple mash (Figure 2). The cellulase promoted a lower viscosity decrease, reducing the viscosity of the apple mash by ∼45% after 5 min maceration (Table 2). The pectinase treatment and the pectinase-cellulase mixture promoted a steeper viscosity decrease of ∼85% and ∼93%, respectively (Table 2). Because of the heterogeneity of apple mash, viscosity measurements may vary significantly, particularly during the first maceration minutes. To compare the different runs, the extent of the viscosity decrease was assessed (Table 2).

Figure 2. Evolution of viscosity of apple mash during 5 min (300 s) of enzyme maceration at 50 °C using individual and combined commercial pectinase and cellulase preparations; data shown as average of two determinations ( SD: ( control, no enzymes added; 2 cellulase treatment; 9 pectinase treatment; • pectinase-cellulase treatment. The data represent a typical set of results and were obtained by a group of students (number of values, n = 3).

Table 1. Specifications for the Commercial Enzyme Preparations Commercial Name

Microbial Producer

Main Activitya

Optimal Reaction Conditions

Pectinex Smash

Aspergillus aculeatus Aspergillus oryzae

Polygalacturonase and pectin methyl esterase 8000 PGU/mL

pH 5, 50 °C

Celluclast 1.5 L

Trichoderma reesei

(Mixed cellulolytic) 700 EGU/g

pH 5, 50 °C

a

Data obtained from Novozymes A/S (Bagsværd, Denmark). PGU: polygalacturonase units; EGU: endoglucanase units.

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In the Laboratory Table 2. Percentage of Viscosity Depletion after Subjecting Apple Mash to Certain Enzyme Preparations for 5 mina Sample

Viscosity depletion (%)

Control

14.2 ( 1.6d

Pectinase

84.3 ( 0.5b

Cellulase

46.5 ( 0.0c

Pectinase-Cellulase

93.0 ( 0.1a

a

Data are shown as average values ( standard deviations. Different superscript letters indicate that values are significantly different at p < 0.05 (pooled standard deviation = 0.856). The data represent a typical set of results and were obtained by a group of students (number of values, n = 3).

The extent of the viscosity determined as ½initial viscosity - viscosity after 5 minutes=½initial viscosity As cellulose and pectin are entangled in the cell-wall network, the degradation of one of these polysaccharides may help the degradation of the other via providing improved access for the enzymes. The data show that simultaneous treatment with pectinase and cellulase results in the highest viscosity decrease, but the effect is not synergistic (Table 2). Depending on the scholarly level and context of the exercise, the issues of synergism among plant cell-wall degrading enzyme activities and calculation of a pooled standard deviation and ANOVA may be discussed. Folin-Ciocalteu Assay Phenol release was measured as soluble phenols present in apple juice after pressing and centrifugation. Because some phenols appear to be entangled in the cellulose-pectin matrix of the cell wall, a higher phenol release is expected when the degree of cell-wall degradation is higher, that is, a positive correlation between viscosity decrease and phenol release. The cellulasepectinase mixture released a higher quantity of phenols than the pectinase treatment alone, but the phenols release obtained with the pectinase treatment was higher than that of the cellulase treatment and the control. However, a true statistically significant difference in the phenol yields was found only between the control and the cellulase-pectinase treatment (Figure 3). These data also lend themselves to a discussion of data evaluation of data sets with varying standard deviations and variable coefficients of variation. A highly positive correlation between phenol release (mg equiv gallic acid/L) and viscosity depletion was found (R2 = 0.965). These findings are in complete accord with the literature on enzymatic treatment of other fruits; with more detailed analyses, it has been confirmed that total phenol release correlates to plant cell-wall polysaccharide degradation (5, 7). Points To Discuss The pectinase contributed to deplete the waterbinding capacity of the apple fruits, hence, decreasing viscosity via depolymerization of the pectin structure forming the middle lamella and part of the primary cell-wall structure of apple. In turn, the phenols entangled in the cell-wall matrix were released. The same behavior was seen with the cellulase treatment, albeit to a much lesser extent. In this exercise, the concept of enzyme unit definition in relation to practical applied enzyme technology can be addressed by discussion. One enzyme unit (katal) is defined as the amount


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Figure 3. Release of phenols from apple mash subjected to maceration using individual and combined commercial pectinase and cellulase preparations. Columns are shown as average values ( standard deviations. Different letters indicate significantly different values at p < 0.05 (pooled standard deviation = 1.22). The data represent a typical set of results and were obtained by a group of students (number of values, n = 3).

of enzyme that will cause the transformation of 1 mol of substrate/s under specified conditions. However, in case of these complex plant polysaccharide substrates, the definition of the molar substrate concentration is not possible and may in fact have no meaning as both pectin and cellulose are polysaccharide structures that require several different enzyme activities to catalyze their full degradation. Hence, although enzyme activity assays can be made using complex substrates, the students can discuss the problems of translating enzyme activity assay data into genuine applications such as this one. Another discussion point could be the relevance of using other cell-wall degrading enzymes that may be envisaged to contribute to enhance the content of phenols in juice, for example, proteases. Proteases are expected to degrade the structural proteins also present in the cell wall. The cell-wall proteins contribute to strengthen the cell-wall structure formed by the polysaccharides. Another point to discuss in class is how to assess the viscosity data obtained (Figure 2); some students will be able to (and attempt to) fit the data to exponential or logarithmic functions, but then may find it difficult to evaluate the statistical significance of the different curves. A simple approach is to compare the averages of the data sets at different time points by ANOVA; this may then lead to a discussion of pooled standard deviation versus set standard deviations and so forth. The results highlight the particular importance of pectinases in apple juice production, and in juice manufacture in general. Pectinases contribute to reduce the viscosity of the fruit mash dramatically, easing the pressing step and enhancing the extraction of certain compounds of interest that would not be extracted otherwise and that may exert beneficial effects on human health. Acknowledgment The results shown in Table 2 and Figures 2 and 3 were obtained by the students of the course Advanced Enzyme Technology during the academic year 2008-2009, as a part of the M.Sc. in Chemical and Biochemical Engineering offered by the Department of Chemical and Biochemical Engineering at the Technical University of Denmark. Notes


1. The EC numbers for the enzymes are pectin methyl esterase (E.C. 3.1.1.11), endopolygalacturonase (E.C. 3.2.1.15), pectin

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In the Laboratory

lyase (E.C. 4.2.2.10), galactanases (E.C. 3.2.1.89 and E.C. 3.2.1.90), arabanases (E.C. 3.2.1.99), and cellulases (E.C. 3.2.1.4, E.C. 3.2.1.74, E.C. 3.2.1.91, and E.C. 3.2.1.21). 2. The laboratory is offered as a part of a MSc-level course at The Technical University of Denmark. 3. Pectinex Smash (pectinase) and Celluclast 1.5 L (cellulase) were used. Both preparations are manufactured by Novozymes A/S, Bagsværd, Denmark.

2. Steinmetz, K. A.; Potter, J. D. Cancer Causes Control 1991, 2, 325– 357. 3. Ness, A. R.; Powles, J. W. Int. J. Epidemiol. 1997, 26, 1–13. 4. Pinelo, M.; Arnous, A.; Meyer, A. S. Trends Food Sci. Technol. 2006, 17, 579–590. 5. Bagger-Jørgensen, R.; Meyer, A. S. Eur. Food Res. Technol. 2004, 219, 620–629. 6. Singleton, V. L.; Rossi, J. A., Jr. Am. J. Enol. Vitic. 1965, 16, 144–158. 7. Arnous, A.; Meyer, A. S. Biochem. Eng. J. 2010, 49, 68–77.

Literature Cited

Supporting Information Available

1. Pinelo, M.; Zornoza, B.; Meyer, A. S. Sep. Purif. Technol. 2008, 63, 620–627.

Student handout. This material is available via the Internet at http://pubs.acs.org.

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A Laboratory Exercise To Understand the Importance of Enzyme

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