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Aspergillus carbonarius polygalacturonases purified by integrated membrane process and affinity precipitation for apple juice production
Bioresource Technology 102 (2011) 3293–3297
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Aspergillus carbonarius polygalacturonases purified by integrated membrane process and affinity precipitation for apple juice production Ekambaram Nakkeeran a,1, Sukumaran Umesh-Kumar b, Rangaswamy Subramanian a,⇑ a b
Department of Food Engineering, Central Food Technological Research Institute, CSIR, Mysore 570 020, India Department of Food Microbiology, Central Food Technological Research Institute, CSIR, Mysore 570 020, India
a r t i c l e
i n f o
Article history: Received 2 June 2010 Received in revised form 8 October 2010 Accepted 11 October 2010 Available online 15 October 2010 Keywords: Aspergillus carbonarius Polygalacturonase Extraction Clarification Apple juice
a b s t r a c t Aspergillus carbonarius, when grown by submerged and solid-state fermentation, produces different molecular forms of polygalacturonase (PG; EC 3.2.1.15), among them a 42 kDa PG with a high specific activity of 7000 U/mg protein. When the enzymes were purified by integrated membrane process (IMP) and alginate affinity precipitation (AAP), the two processes concentrated different forms of the enzyme. The AAP process selectively purified and concentrated the high active PG whereas the IMP yielded different PGs and also amylase and protease. Evaluation of the AAP enzyme preparations for apple juice preparation under conditions usually employed commercially demonstrated that the high activity PG did not result in good juice clarity. With IMP processed enzymes, juice yields and clarity were similar to that obtained with commercial PG from A. niger. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Pectinases, specifically polygalacturonases (PG), find applications in the paper and textile industry, and in food processing especially in the production of fruit juice (Kashyap et al., 2001). Pectinases from microbial sources make up almost 25% of global food enzyme sales (Tari et al., 2007). Apple juice is the most popular juice worldwide next only to orange juice (Kahle et al., 2005). For the production of apple juice concentrate, enzymatic treatment of crushed apple mash with pectinase followed by pomace extraction is widely practiced (Faigh, 1995). PGs that catalyze the degradation of pectin polymers lower viscosity and water binding capacity for easier juice extraction and better working capacity of presses or decanters (Will et al., 2002). Commercial pectinases, predominantly containing PGs, have been used in apple juice preparation for higher juice yield, clarity, colloid concentration and polyphenolic contents (Faigh, 1995; Will et al., 2002; Sorrivas et al., 2006; Oszmianski et al., 2009). Commercial apple juices treated with15 U pectinase of Aspergillus niger van Tieghem per ml of juice in the presence of 0.01% gelatin at 45 °C for 6 h provided a clear juice (%T650, 85%) along with a viscosity drop of 35.5% (Singh and Gupta, 2004). PGs of other fungi, bacteria and yeasts have also been explored. For example, Kaur et al. (2004) reported an increase ⇑ Corresponding author. Tel.: +91 821 251 3910; fax: +91 821 251 7233. E-mail address: [email protected] (R. Subramanian). Present address: Industrial Biotechnology Division, School of Bio Sciences and Technology, VIT University, Vellore 632 014, India. 1
0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.10.048
in juice yield when apple, banana and grapes were treated with Sporotrichum thermophile PG. Semenova et al. (2006) tested the efficacy of chromatographically purified Penicillium canescens pectin lyase for the clarification of commercial non-clarified apple juice, and reported that its performance was comparable with that of commercial pectinase preparations. Bacillus pumilus pectin lyase has also been studied for the extraction of various fruit juices (Nadarog˘lu et al., 2010). Some of the enzymes in commercial pectinase preparations containing A. niger PGs (de Vries and Visser, 2001) have been purified and characterized. The A. niger PGs exhibited specific activities ranging from 25–4000 U/mg protein. Four of these PGs showed multiple attacks on a single chain and three of them exhibited a single attack (Perinicova et al., 2000a,b). Apple pectin contains branched homogalacturonans carrying various amounts of b-D-xylose (Wong, 2008). Commercial pectinase of A. niger also contained PG with activity towards xylogalacturonans (Sakamoto et al., 2002). Thus activity of multiple PGs seems important for fruit juice extraction and clarification. Like A. niger, A. carbonarius (a food safe fungus) also secretes multiple PGs, among them an 42 kDa enzyme with a specific activity of 7000 U/mg protein (Devi and Appu Rao, 1996) that is serologically related to PGs produced by A. niger (Venkatesh and Umesh-Kumar, 2005). It is currently not known if this high activity enzyme would be useful for apple juice production. Industrially, microbial PGs are produced by solid-state (SSF) and submerged fermentation (SmF) processes (Naidu and Panda, 1998; Semenova et al., 2006). Venkatesh (2004) showed that
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A. carbonarius produced different enzymes in SSF and SmF. The fungus secreted the highly active 42 kDa PG in SmF while in SSF, the predominant PGs were 61 and 47 kDa proteins. Integrated membrane process (IMP) developed for the purification of SmF-PG, increased the purity 4.7-fold (Nakkeeran et al., 2008), but did not improve the purity of SSF-PG owing to the presence of other similar molecular mass proteins in the extracts (Nakkeeran et al., 2010). Alginate affinity precipitation (AAP) enhanced the purity of SmF and SSF-PG, and showed selectivity for the 42 kDa PG (Nakkeeran et al., 2009). In this study, the activities of IMP and AAP purified enzyme preparations from A. carbonarius were compared with A. niger commercial pectinase, obtained from SPIC and Sigma, for extraction and clarification of apple juice. The details are described in this paper. 2. Methods 2.1. Chemicals, enzymes and raw materials Galacturonic acid was procured from M/s Sigma Chemicals, St. Louis, USA. Other analytical and laboratory grade chemicals were purchased from M/s Hi-Media Laboratories or M/s Sisco Research Laboratories, Mumbai, India. A. carbonarius PG, produced in submerged and solid-state cultures, were purified using integrated membrane process and alginate affinity purification as described earlier (Nakkeeran et al., 2009). Commercial pectinase preparations were obtained from M/s SPIC Biotechnology Division, Chennai, India (commercial PG-1; 1620 U/ml; A. niger) and M/s Sigma–aldrich, Bangalore, India (commercial PG-2; 570 U/ml; A. niger). Apples (Ambri Kashmir variety) of proper maturity and ripeness were purchased from the local market. 2.2. Enzymatic extraction and clarification of apple juice Apples purchased from local market were washed, peeled and cut into small pieces. Ascorbic acid (0.5 g/kg) was added and the cut apples were mashed using a food processor for 30 s. To the mash, appropriate quantities of enzyme were added and extraction was carried out at 30 ± 2 °C and 52 ± 2 °C for 2 and 4 h, respectively. The treated mash was heated to 90 °C for 5 min in a water bath to inactivate the enzymes. Juice from the mash was separated by centrifugation at 2200g for 10 min and pomace was discarded. 2.3. Polygalacturonase assay PG activity was determined using 0.5% polygalacturonic acid (sodium salt) prepared in 0.1 M sodium acetate buffer (pH 4.3) as substrate. Assays were carried out for 10 min at 50 °C (Nakkeeran et al., 2010) and the reducing sugars were quantified as galacturonic acid equivalents (Somogyi, 1952). Activity (ml1) corresponded to micromole galacturonic acid released per minute per milliliter. 2.4. Protease assay Protease activity was determined using 2% casein (Hammarsten). The substrate was dissolved in distilled water by boiling for 15 min. After cooling, the pH was adjusted to 2.7 using 0.1 N HCl. Assays were carried out for 30 min at 30 °C and the released tyrosine equivalent was estimated using Folin–Ciocalteau phenol reagent (Ichishima, 1970). Activity (ml1) corresponded to micromole tyrosine released per minute per milliliter.
ried out for 30 min at 40 °C and the reducing sugars were quantified as maltose equivalents (Bernfeld, 1955). Activity (ml1) corresponded to micromole maltose released per minute per milliliter. 2.6. Protein estimation Protein content was determined by the dye binding method (Spector, 1978) using Coomassie Brilliant Blue G 250. Bovine serum albumin was used as a standard. 2.7. Estimation of juice yield Juice yield was determined using the following equation.
Juice yield ð%Þ ¼
Final juice weight 100 Initial mash weight
2.8. Clarity and turbidity measurements Clarity of juice was measured according to the method of Krop and Pilnik (1974). Percent transmittance was determined at 660 nm using a spectrophotometer (UV-160A, M/s Shimadzu Scientific Instruments, Kyoto, Japan). Turbidity of juices was measured using a benchtop turbidity meter (Model: Cyberscan Turbidimeter TB1000, M/s Eutech Instruments Pte Ltd., Singapore) and expressed in nephelometric turbidity unit (NTU). 2.9. Estimation of alcohol-insoluble-solids The content of pectinnious materials present in the apple juices was measured in terms of alcohol-insoluble-solids (AIS). AIS were determined by boiling 5 g juice with 75 ml 80% ethanol, simmering for 30 min, and filtering through Whatman No. 1 filter paper. The filtered residue was washed again with 80% ethanol. The residue was dried at 100 °C for 2 h and expressed in percentage by weight (Hart and Fisher, 1971). 2.10. Hunter colour values Colour of juice was measured using a Hunter Lab Colour Measuring System (Model: Labscan XE, M/s Hunter Associates Laboratory Inc., Reston, USA) at 2°/C angle of illumination. Values were measured in terms of lightness (L) and colour (+a: red, a: green, +b: yellow, b: blue). 2.11. Determination of total soluble solids, viscosity and density Total soluble solids (TSS) were measured using a hand held refractometer (Model: RHB-32(ATC), M/s HTA Instrumentation, Bangalore, India) and expressed as °Brix. Viscosity was measured using a glass Oswald capillary viscometer (M/s Borosil Glass Work Ltd., Mumbai, India) at a constant water bath temperature of 30 ± 2 °C. The density of juice was determined using a specific gravity bottle (M/s Borosil Glass Work Ltd.). 2.12. pH measurement pH was measured using a digital glass electrode pH meter (M/s Control Dynamics, Bangalore, India). It was calibrated using buffer solutions of pH 4.0 and 7.0 at room temperature (28 ± 2 °C).
2.5. Amylase assay
2.13. Statistical analysis
Amylase activity was determined using 2% starch prepared in 0.1 M sodium acetate buffer (pH 4.3) as substrate. Assays were car-
The data were analyzed using one-way analysis of variance (ANOVA) and means for each pair were compared at 5%
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significance level by using t-test, in Microsoft Excel Data Analysis Tool Pack. 3. Results and discussion Enzymatic treatments of fruit juice are carried out at 54oC (hot treatment) or at lower temperatures (cold treatment) (Oke and Paliyath, 2006). Since PGs are the major constituents for the fruit juice production process, their activities under the above conditions were compared for apple juice preparation. Higher juice yield and clarity were obtained with commercial pectinases at an elevated temperature of 50 °C, compared to 30 °C (Table 1). Increased sugar yield at higher incubation temperature was also obtained when apple mash was processed with A. carbonarius PGs (Fig. 1). Apple mash processed using AAP-PGs showed more turbidity
(219–238 NTU) compared to higher clarity juice (turbidity, 96–102 NTU) obtained with SSF crude PG, SSF-IMP-PG and SmFIMP-PG which was closer to the 56–76 NTU values obtained with the commercial PGs (Fig. 2). Apple mash not treated to enzymes (control) resulted in low sugar yield and clarity. Mash treated with commercial PGs and crude and purified PGs of A. carbonarius resulted in higher juice yield, sugar recovery and clarity (Table 2). The sugar yields obtained with IMP processed PGs were not significantly different from those obtained with commercial PGs. In contrast, yields of sugar obtained with AAP processed PGs were significantly lower. Juice yields paralleled sugar yields and the differences among TSS values were only marginal with various PGs. AAP exhibited a high selectivity for the highly active 42 kDa PG, secreted as the predominant PG in SmF (Venkatesh, 2004). The
Table 1 Enzymatic extraction and clarification of apple juice using commercial PG-1A. PG addition (U/g mash)
Temperature (°C)
Period (h)
Juice yield (%)
Clarity (%T at 660 nm)
TSS (°Brix)
pH
Control
30
2 4 2 4 2 4 2 4 2 4 2 4 2 4 2 4
15.6 ± 1.2a 20.8 ± 1.1a 17.3 ± 1.3a 22.4 ± 1.9a 43.9 ± 0.7a 55.2 ± 0.5b 57.8 ± 1.4a 60.2 ± 2.0b 61.8 ± 0.8a 66.7 ± 0.6a 67.3 ± 0.5a 68.2 ± 1.0a 69.0 ± 1.3a 70.7 ± 1.5a 78.5 ± 1.6a 81.8 ± 1.3a
0.9 ± 0.2a 1.5 ± 0.3a 3.1 ± 1.0a 4.3 ± 1.1a 29.2 ± 1.1a 46.5 ± 0.9b 57.0 ± 1.0a 62.1 ± 1.4a 52.0 ± 1.3a 62.7 ± 1.2b 69.8 ± 0.6a 74.2 ± 1.4a 70.2 ± 1.2a 80.3 ± 0.9b 83.5 ± 0.6a 85.2 ± 1.4a
11.2 11.4 11.4 11.8 11.8 11.8 11.8 12.0 12.0 12.0 11.8 12.2 12.2 12.2 12.2 12.4
3.72 3.63 3.68 3.64 3.64 3.63 3.64 3.64 3.53 3.49 3.50 3.48 3.40 3.38 3.36 3.32
50 0.5
30 50
1.25
30 50
2.5
30 50
ab Means along a column at a particular PG addition and temperature with different duration having different superscripts are significantly different at p < 0.05 (n = 3). A PG, Polygalacturonase; TSS, Total soluble solids.
Fig. 1. Sugar yield from apple mash treated to commercial and A. carbonarius PGs. Apple mashes were treated, for 2 h, with commercial PGs (com-1 & com-2) or A. carbonarius PGs obtained after submerged (SmF) and solid- state (SSF) fermentation. Crude (C), enzyme purified by alginate affinity precipitation (AAP) and integrated membrane process (IMP) were used for the experiment. Control: No enzyme treatment.
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Fig. 2. Turbidity of apple juice prepared using commercial and A. carbonarius PGs. Apple juice was prepared with commercial PGs (com-1 & com-2) or A. carbonarius PGs obtained after submerged (SmF) and solid-state (SSF) fermentation. Crude (C), enzyme purified by alginate affinity precipitation (AAP) and integrated membrane process (IMP) were used for the experiment. Control: No enzyme treatment.
Table 2 Enzymatic extraction and clarification of apple juice using various PG samplesA. Type of PG
Control SmF-Crude SSF-Crude SmF-IMP SSF-IMP SmF-AAP SSF-AAP Commercial-1 Commercial-2
Juice yield (%)
20.5 ± 0.5a 74.1 ± 1.2bfghi 76.0 ± 1.0bcfghi 73.9 ± 1.2bdfghi 76.7 ± 1.2befghi 72.8 ± 0.9fg 73.6 ± 1.3g 81.6 ± 1.1hi 83.1 ± 0.8i
Turbidity (NTU)
Colour L
a
b
1026 173 99 102 96 238 219 76 56
35.46 43.69 43.53 48.45 48.32 38.79 42.26 45.03 49.56
8.50 3.55 2.87 0.44 1.44 6.88 4.42 2.72 0.22
32.52 34.30 32.41 28.11 31.93 38.93 36.79 34.12 27.12
TSS (°Brix)
pH
Density ( 103 kg/m3)
Viscosity (m Pa s)
AIS (wt.%)
Sugar yield (%)
14.2 14.6 14.6 14.6 14.6 14.4 14.6 14.8 14.8
4.05 3.82 3.81 3.82 3.82 3.85 3.84 3.61 3.55
1.10 1.09 1.09 1.09 1.09 1.09 1.09 1.10 1.10
3.33 1.27 1.20 1.27 1.20 1.27 1.27 1.13 1.13
1.12 0.58 0.56 0.54 0.56 0.60 0.62 0.46 0.50
2.9 ± 0.1a 10.8 ± 0.2bfghi 11.1 ± 0.1bcfghi 10.8 ± 0.2bdfghi 11.2 ± 0.2befghi 10.5 ± 0.1fg 10.7 ± 0.2g 12.1 ± 0.2hi 12.3 ± 0.1i
PG addition – 5 U/g mash; Incubation at 50 °C for 2 h. a–i Means along a column with different superscripts are significantly different at p < 0.05 (n = 3). A NTU, Nephelometric turbidity units; AIS, Alcohol insoluble solids; SmF, Submerged fermentation; SSF, Solid-state fermentation; IMP, Integrated membrane process; AAP, Alginate affinity purification. For other abbreviations see Table 1.
process almost completely eliminated other PGs and contaminant proteins in the SmF- and SSF-cultures (Nakkeeran et al., 2009). Since apple mash processed using AAP-PGs showed more turbidity, the highly active PG of A. carbonarius would not be attractive for commercial application. IMP employing microfiltration (MF) and ultrafiltration (UF) membranes retained all the PGs. The process did not eliminate protease and amylase, since they were similar in molecular mass as that of PG (Nakkeeran et al., 2009, 2010). The presence of these enzymes in IMP processed PG preparations (Table 3) apparently further aided cell wall hydrolysis sufficient to obtain a performance comparable to those of the A. niger commercial enzymes. A reduction in viscosity of juice by about 66% was observed with the PGs after enzyme treatment of mash. Singh and Gupta (2004) reported a viscosity drop of 36% during the enzymatic clarification of commercial apple juice and Busto et al. (2006) reported a reduction of 35% during enzymatic treatment of apple pectin, but reductions in juice viscosity as high as 82–91% have been reported during enzymatic treatment of apple mash with different commercial pectolytic enzymes (Oszmianski et al., 2009).
Table 3 Enzyme activities of various PG samplesA.
A
Type of PG
PG (U/g mash)
Protease ( 103 U/g mash)
Amylase ( 102 U/g mash)
PG specific activity (U/mg protein)
SmF-Crude SSF-Crude SmF-IMP SSF-IMP SmF-AAP SSF-AAP Commercial-1 Commercial-2
5 5 5 5 5 5 5 5
0.4 2.6 0.8 5.8 ND ND 3.5 4.1
0.7 4.1 0.9 4.6 ND ND 37 44
1190 530 5590 640 9770 2450 330 120
ND, Not detected; For other abbreviations see Tables 1 and 2.
The differences in performances could be ascribed to many factors, such as type of enzyme preparation, apple variety, treatment and pressing conditions. There was no considerable difference in pH, colour and density of juice obtained from treated apple mashes with various PGs (Table 2).
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4. Conclusions Though A. carbonarius produces a high active PG, the enzyme alone would not be suitable for industrial application. Only the IMP-derived enzyme preparation which also contained protease and amylase produced apple juice comparable to that produced by commercial A. niger enzyme preparations. Acknowledgements E. Nakkeeran thanks CSIR, New Delhi, India, for the award of fellowship. P. Vijayanand and N. Kumaresan at CFTRI provided valuable advice. References Bernfeld, P., 1955. Amylases alpha and beta. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology. Academic press, New York, pp. 149–150. Busto, M.D., García-Tramontín, K.E., Ortega, N., Perez-Mateos, M., 2006. Preparation and properties of an immobilized pectinlyase for the treatment of fruit juices. Bioresour. Technol. 97, 1477–1483. de Vries, R.P., Visser, J., 2001. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiol. Mol. Biol. Rev. 65, 497–522. Devi, N.A., Appu Rao, A.G., 1996. Fractionation, purification, and preliminary characterization of polygalacturonases produced by Aspergillus carbonarius. Enzyme Microb. Technol. 18, 59–65. Faigh, J.G., 1995. Enzyme formulations for optimizing juice yields. Food Technol. 49, 79–83. Hart, F.L., Fisher, H.J., 1971. Modern Food Analysis. Springer, Berlin. Ichishima, E., 1970. Purification and mode of assay for acid proteinase of Aspergillus saitoi. In: Perlmann, G.E., Lorand, L. (Eds.), Methods in Enzymology. Academic press, New York, pp. 397–406. Kahle, K., Kraus, M., Richling, E., 2005. Polyphenol profiles of apple juices. Mol. Nutr. Food Res. 49, 797–806. Kashyap, D.R., Vohra, P.K., Chopra, S., Tewari, R., 2001. Applications of pectinases in the commercial sector: a review. Bioresour. Technol. 77, 215–227. Kaur, G., Kumar, S., Satyanarayana, T., 2004. Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis. Bioresour. Technol. 94, 239–243. Krop, J.J.P., Pilnik, W., 1974. Effect of pectic acid and bivalent cations on cloud loss of citrus juice. LWT 7, 62–73. Nadarog˘lu, H., Tasßkin, E., Adigüzel, A., Güllüce, M., Demir, N., 2010. Production of a novel pectin lyase from Bacillus pumilus (P9), purification and characterisation and fruit juice application. Romanian Biotechnol. Lett. 15, 5167–5176.
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Naidu, G.S.N., Panda, T., 1998. Production of pectolytic enzymes - a review. Bioproc. Eng. 19, 355–361. Nakkeeran, E., Subramanian, R., Umesh Kumar, S., 2008. Improving specific activity of Aspergillus carbonarius polygalacturonase using polymeric membranes. Appl. Biochem. Biotechnol. 151, 233–243. Nakkeeran, E., Subramanian, R., Umesh Kumar, S., 2009. Process assessment for the purification of Aspergillus carbonarius polygalacturonase produced by submerged and solid-state fermentations. Int. J. Eng. Technol. 4, 278–281. Nakkeeran, E., Subramanian, R., Umesh Kumar, S., 2010. Purification of polygalacturonase from solid-state cultures of Aspergillus carbonarius. J. Biosci. Bioeng. 109, 101–106. Oke, M., Paliyath, G., 2006. Biochemistry of fruit processing. In: Hui, Y.H. (Ed.), Food Biochemistry. Wiley-Blackwell Publishing, Massachusetts, pp. 515–536. Oszmianski, J., Wojdylo, A., Kolniak, J., 2009. Effect of enzymatic mash treatment and storage on phenolic composition, antioxidant activity, and turbidity of cloudy apple juice. J. Agric. Food Chem. 57, 7078–7085. Perinicova, L., Benen, J.A.E., Kester, H.C.M., Visser, J., 2000a. PgaA and PgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger. Biochem. J. 345, 637–644. Perinicova, L., Kester, H.C.M., Benen, J.A.E., Visser, J., 2000b. Characterization of a novel endopolygalacturonase from Aspergillus niger with unique kinetic properties. FEBS Lett. 467, 333–336. Sakamoto, T., Bonnin, E., Quemener, B., Thiabault, J.-F., 2002. Purification and characterisation of two exo-polygalacturonases from Aspergillus niger able to degrade xylogalacturonan and acetylated homogalacturonan. Biochem. Biophys. Acta 1572, 10–18. Semenova, M.V., Sinitsyna, O.A., Morozova, V.V., Fedorova, E.A., Gusakov, A.V., Okunev, O.N., Sokolova, L.M., Koshelev, A.V., Bubnova, T.V., Vinetskii, Yu.P., Sinitsyn, A.P., 2006. Use of a preparation from fungal pectin lyase in the food industry. Appl. Biochem. Microbiol. 42, 598–602. Singh, S., Gupta, R., 2004. Apple juice clarification using fungal pectinolytic enzyme and gelatin. Indian J. Biotechnol. 3, 573–576. Somogyi, M., 1952. Notes on sugar determination. J. Biol. Chem. 195, 19–23. Sorrivas, V., Genovese, D.B., Lozano, J.E., 2006. Effect of pectinolytic and amylolytic enzymes on apple juice turbidity. J. Food Proc. Preserv. 30, 118–133. Spector, T., 1978. Refinement of coomassie blue method of protein quantitation. Anal. Biochem. 86, 142–146. Tari, C., Gogus, N., Tokatli, F., 2007. Optimization of biomass, pellet size and polygalacturonase production by Aspergillus sojae ATCC 20235 using response surface methodology. Enzyme Microb. Technol. 40, 1108–1116. Venkatesh, K.S., 2004. Strain and process improvement for polygalacturonase production by Aspergillus carbonarius, Ph. D. Thesis, University of Mysore, India. Venkatesh, K.S., Umesh-Kumar, S., 2005. Production of pectinases and utilization in food processing. In: Shetty, K., Paliyath, G., Pometto, A., Levin, R.E. (Eds.), Food Biotechnol. CRC press, New York, pp. 329–348. Will, F., Schulz, K., Ludwig, M., Otto, K., Dietrich, H., 2002. The influence of enzymatic treatment of mash on the analytical composition of apple juice. Int. J. Food Sci. Technol. 37, 653–660. Wong, D., 2008. Enzymatic destruction of backbone structures of the ramified regions in pectins. Protein J. 27, 37–42.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Aspergillus carbonarius polygalacturonases purified by integrated membrane process and affinity precipitation for apple juice production Ekambaram Nakkeeran a,1, Sukumaran Umesh-Kumar b, Rangaswamy Subramanian a,⇑ a b
Department of Food Engineering, Central Food Technological Research Institute, CSIR, Mysore 570 020, India Department of Food Microbiology, Central Food Technological Research Institute, CSIR, Mysore 570 020, India
a r t i c l e
i n f o
Article history: Received 2 June 2010 Received in revised form 8 October 2010 Accepted 11 October 2010 Available online 15 October 2010 Keywords: Aspergillus carbonarius Polygalacturonase Extraction Clarification Apple juice
a b s t r a c t Aspergillus carbonarius, when grown by submerged and solid-state fermentation, produces different molecular forms of polygalacturonase (PG; EC 3.2.1.15), among them a 42 kDa PG with a high specific activity of 7000 U/mg protein. When the enzymes were purified by integrated membrane process (IMP) and alginate affinity precipitation (AAP), the two processes concentrated different forms of the enzyme. The AAP process selectively purified and concentrated the high active PG whereas the IMP yielded different PGs and also amylase and protease. Evaluation of the AAP enzyme preparations for apple juice preparation under conditions usually employed commercially demonstrated that the high activity PG did not result in good juice clarity. With IMP processed enzymes, juice yields and clarity were similar to that obtained with commercial PG from A. niger. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Pectinases, specifically polygalacturonases (PG), find applications in the paper and textile industry, and in food processing especially in the production of fruit juice (Kashyap et al., 2001). Pectinases from microbial sources make up almost 25% of global food enzyme sales (Tari et al., 2007). Apple juice is the most popular juice worldwide next only to orange juice (Kahle et al., 2005). For the production of apple juice concentrate, enzymatic treatment of crushed apple mash with pectinase followed by pomace extraction is widely practiced (Faigh, 1995). PGs that catalyze the degradation of pectin polymers lower viscosity and water binding capacity for easier juice extraction and better working capacity of presses or decanters (Will et al., 2002). Commercial pectinases, predominantly containing PGs, have been used in apple juice preparation for higher juice yield, clarity, colloid concentration and polyphenolic contents (Faigh, 1995; Will et al., 2002; Sorrivas et al., 2006; Oszmianski et al., 2009). Commercial apple juices treated with15 U pectinase of Aspergillus niger van Tieghem per ml of juice in the presence of 0.01% gelatin at 45 °C for 6 h provided a clear juice (%T650, 85%) along with a viscosity drop of 35.5% (Singh and Gupta, 2004). PGs of other fungi, bacteria and yeasts have also been explored. For example, Kaur et al. (2004) reported an increase ⇑ Corresponding author. Tel.: +91 821 251 3910; fax: +91 821 251 7233. E-mail address: [email protected] (R. Subramanian). Present address: Industrial Biotechnology Division, School of Bio Sciences and Technology, VIT University, Vellore 632 014, India. 1
0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.10.048
in juice yield when apple, banana and grapes were treated with Sporotrichum thermophile PG. Semenova et al. (2006) tested the efficacy of chromatographically purified Penicillium canescens pectin lyase for the clarification of commercial non-clarified apple juice, and reported that its performance was comparable with that of commercial pectinase preparations. Bacillus pumilus pectin lyase has also been studied for the extraction of various fruit juices (Nadarog˘lu et al., 2010). Some of the enzymes in commercial pectinase preparations containing A. niger PGs (de Vries and Visser, 2001) have been purified and characterized. The A. niger PGs exhibited specific activities ranging from 25–4000 U/mg protein. Four of these PGs showed multiple attacks on a single chain and three of them exhibited a single attack (Perinicova et al., 2000a,b). Apple pectin contains branched homogalacturonans carrying various amounts of b-D-xylose (Wong, 2008). Commercial pectinase of A. niger also contained PG with activity towards xylogalacturonans (Sakamoto et al., 2002). Thus activity of multiple PGs seems important for fruit juice extraction and clarification. Like A. niger, A. carbonarius (a food safe fungus) also secretes multiple PGs, among them an 42 kDa enzyme with a specific activity of 7000 U/mg protein (Devi and Appu Rao, 1996) that is serologically related to PGs produced by A. niger (Venkatesh and Umesh-Kumar, 2005). It is currently not known if this high activity enzyme would be useful for apple juice production. Industrially, microbial PGs are produced by solid-state (SSF) and submerged fermentation (SmF) processes (Naidu and Panda, 1998; Semenova et al., 2006). Venkatesh (2004) showed that
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A. carbonarius produced different enzymes in SSF and SmF. The fungus secreted the highly active 42 kDa PG in SmF while in SSF, the predominant PGs were 61 and 47 kDa proteins. Integrated membrane process (IMP) developed for the purification of SmF-PG, increased the purity 4.7-fold (Nakkeeran et al., 2008), but did not improve the purity of SSF-PG owing to the presence of other similar molecular mass proteins in the extracts (Nakkeeran et al., 2010). Alginate affinity precipitation (AAP) enhanced the purity of SmF and SSF-PG, and showed selectivity for the 42 kDa PG (Nakkeeran et al., 2009). In this study, the activities of IMP and AAP purified enzyme preparations from A. carbonarius were compared with A. niger commercial pectinase, obtained from SPIC and Sigma, for extraction and clarification of apple juice. The details are described in this paper. 2. Methods 2.1. Chemicals, enzymes and raw materials Galacturonic acid was procured from M/s Sigma Chemicals, St. Louis, USA. Other analytical and laboratory grade chemicals were purchased from M/s Hi-Media Laboratories or M/s Sisco Research Laboratories, Mumbai, India. A. carbonarius PG, produced in submerged and solid-state cultures, were purified using integrated membrane process and alginate affinity purification as described earlier (Nakkeeran et al., 2009). Commercial pectinase preparations were obtained from M/s SPIC Biotechnology Division, Chennai, India (commercial PG-1; 1620 U/ml; A. niger) and M/s Sigma–aldrich, Bangalore, India (commercial PG-2; 570 U/ml; A. niger). Apples (Ambri Kashmir variety) of proper maturity and ripeness were purchased from the local market. 2.2. Enzymatic extraction and clarification of apple juice Apples purchased from local market were washed, peeled and cut into small pieces. Ascorbic acid (0.5 g/kg) was added and the cut apples were mashed using a food processor for 30 s. To the mash, appropriate quantities of enzyme were added and extraction was carried out at 30 ± 2 °C and 52 ± 2 °C for 2 and 4 h, respectively. The treated mash was heated to 90 °C for 5 min in a water bath to inactivate the enzymes. Juice from the mash was separated by centrifugation at 2200g for 10 min and pomace was discarded. 2.3. Polygalacturonase assay PG activity was determined using 0.5% polygalacturonic acid (sodium salt) prepared in 0.1 M sodium acetate buffer (pH 4.3) as substrate. Assays were carried out for 10 min at 50 °C (Nakkeeran et al., 2010) and the reducing sugars were quantified as galacturonic acid equivalents (Somogyi, 1952). Activity (ml1) corresponded to micromole galacturonic acid released per minute per milliliter. 2.4. Protease assay Protease activity was determined using 2% casein (Hammarsten). The substrate was dissolved in distilled water by boiling for 15 min. After cooling, the pH was adjusted to 2.7 using 0.1 N HCl. Assays were carried out for 30 min at 30 °C and the released tyrosine equivalent was estimated using Folin–Ciocalteau phenol reagent (Ichishima, 1970). Activity (ml1) corresponded to micromole tyrosine released per minute per milliliter.
ried out for 30 min at 40 °C and the reducing sugars were quantified as maltose equivalents (Bernfeld, 1955). Activity (ml1) corresponded to micromole maltose released per minute per milliliter. 2.6. Protein estimation Protein content was determined by the dye binding method (Spector, 1978) using Coomassie Brilliant Blue G 250. Bovine serum albumin was used as a standard. 2.7. Estimation of juice yield Juice yield was determined using the following equation.
Juice yield ð%Þ ¼
Final juice weight 100 Initial mash weight
2.8. Clarity and turbidity measurements Clarity of juice was measured according to the method of Krop and Pilnik (1974). Percent transmittance was determined at 660 nm using a spectrophotometer (UV-160A, M/s Shimadzu Scientific Instruments, Kyoto, Japan). Turbidity of juices was measured using a benchtop turbidity meter (Model: Cyberscan Turbidimeter TB1000, M/s Eutech Instruments Pte Ltd., Singapore) and expressed in nephelometric turbidity unit (NTU). 2.9. Estimation of alcohol-insoluble-solids The content of pectinnious materials present in the apple juices was measured in terms of alcohol-insoluble-solids (AIS). AIS were determined by boiling 5 g juice with 75 ml 80% ethanol, simmering for 30 min, and filtering through Whatman No. 1 filter paper. The filtered residue was washed again with 80% ethanol. The residue was dried at 100 °C for 2 h and expressed in percentage by weight (Hart and Fisher, 1971). 2.10. Hunter colour values Colour of juice was measured using a Hunter Lab Colour Measuring System (Model: Labscan XE, M/s Hunter Associates Laboratory Inc., Reston, USA) at 2°/C angle of illumination. Values were measured in terms of lightness (L) and colour (+a: red, a: green, +b: yellow, b: blue). 2.11. Determination of total soluble solids, viscosity and density Total soluble solids (TSS) were measured using a hand held refractometer (Model: RHB-32(ATC), M/s HTA Instrumentation, Bangalore, India) and expressed as °Brix. Viscosity was measured using a glass Oswald capillary viscometer (M/s Borosil Glass Work Ltd., Mumbai, India) at a constant water bath temperature of 30 ± 2 °C. The density of juice was determined using a specific gravity bottle (M/s Borosil Glass Work Ltd.). 2.12. pH measurement pH was measured using a digital glass electrode pH meter (M/s Control Dynamics, Bangalore, India). It was calibrated using buffer solutions of pH 4.0 and 7.0 at room temperature (28 ± 2 °C).
2.5. Amylase assay
2.13. Statistical analysis
Amylase activity was determined using 2% starch prepared in 0.1 M sodium acetate buffer (pH 4.3) as substrate. Assays were car-
The data were analyzed using one-way analysis of variance (ANOVA) and means for each pair were compared at 5%
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significance level by using t-test, in Microsoft Excel Data Analysis Tool Pack. 3. Results and discussion Enzymatic treatments of fruit juice are carried out at 54oC (hot treatment) or at lower temperatures (cold treatment) (Oke and Paliyath, 2006). Since PGs are the major constituents for the fruit juice production process, their activities under the above conditions were compared for apple juice preparation. Higher juice yield and clarity were obtained with commercial pectinases at an elevated temperature of 50 °C, compared to 30 °C (Table 1). Increased sugar yield at higher incubation temperature was also obtained when apple mash was processed with A. carbonarius PGs (Fig. 1). Apple mash processed using AAP-PGs showed more turbidity
(219–238 NTU) compared to higher clarity juice (turbidity, 96–102 NTU) obtained with SSF crude PG, SSF-IMP-PG and SmFIMP-PG which was closer to the 56–76 NTU values obtained with the commercial PGs (Fig. 2). Apple mash not treated to enzymes (control) resulted in low sugar yield and clarity. Mash treated with commercial PGs and crude and purified PGs of A. carbonarius resulted in higher juice yield, sugar recovery and clarity (Table 2). The sugar yields obtained with IMP processed PGs were not significantly different from those obtained with commercial PGs. In contrast, yields of sugar obtained with AAP processed PGs were significantly lower. Juice yields paralleled sugar yields and the differences among TSS values were only marginal with various PGs. AAP exhibited a high selectivity for the highly active 42 kDa PG, secreted as the predominant PG in SmF (Venkatesh, 2004). The
Table 1 Enzymatic extraction and clarification of apple juice using commercial PG-1A. PG addition (U/g mash)
Temperature (°C)
Period (h)
Juice yield (%)
Clarity (%T at 660 nm)
TSS (°Brix)
pH
Control
30
2 4 2 4 2 4 2 4 2 4 2 4 2 4 2 4
15.6 ± 1.2a 20.8 ± 1.1a 17.3 ± 1.3a 22.4 ± 1.9a 43.9 ± 0.7a 55.2 ± 0.5b 57.8 ± 1.4a 60.2 ± 2.0b 61.8 ± 0.8a 66.7 ± 0.6a 67.3 ± 0.5a 68.2 ± 1.0a 69.0 ± 1.3a 70.7 ± 1.5a 78.5 ± 1.6a 81.8 ± 1.3a
0.9 ± 0.2a 1.5 ± 0.3a 3.1 ± 1.0a 4.3 ± 1.1a 29.2 ± 1.1a 46.5 ± 0.9b 57.0 ± 1.0a 62.1 ± 1.4a 52.0 ± 1.3a 62.7 ± 1.2b 69.8 ± 0.6a 74.2 ± 1.4a 70.2 ± 1.2a 80.3 ± 0.9b 83.5 ± 0.6a 85.2 ± 1.4a
11.2 11.4 11.4 11.8 11.8 11.8 11.8 12.0 12.0 12.0 11.8 12.2 12.2 12.2 12.2 12.4
3.72 3.63 3.68 3.64 3.64 3.63 3.64 3.64 3.53 3.49 3.50 3.48 3.40 3.38 3.36 3.32
50 0.5
30 50
1.25
30 50
2.5
30 50
ab Means along a column at a particular PG addition and temperature with different duration having different superscripts are significantly different at p < 0.05 (n = 3). A PG, Polygalacturonase; TSS, Total soluble solids.
Fig. 1. Sugar yield from apple mash treated to commercial and A. carbonarius PGs. Apple mashes were treated, for 2 h, with commercial PGs (com-1 & com-2) or A. carbonarius PGs obtained after submerged (SmF) and solid- state (SSF) fermentation. Crude (C), enzyme purified by alginate affinity precipitation (AAP) and integrated membrane process (IMP) were used for the experiment. Control: No enzyme treatment.
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Fig. 2. Turbidity of apple juice prepared using commercial and A. carbonarius PGs. Apple juice was prepared with commercial PGs (com-1 & com-2) or A. carbonarius PGs obtained after submerged (SmF) and solid-state (SSF) fermentation. Crude (C), enzyme purified by alginate affinity precipitation (AAP) and integrated membrane process (IMP) were used for the experiment. Control: No enzyme treatment.
Table 2 Enzymatic extraction and clarification of apple juice using various PG samplesA. Type of PG
Control SmF-Crude SSF-Crude SmF-IMP SSF-IMP SmF-AAP SSF-AAP Commercial-1 Commercial-2
Juice yield (%)
20.5 ± 0.5a 74.1 ± 1.2bfghi 76.0 ± 1.0bcfghi 73.9 ± 1.2bdfghi 76.7 ± 1.2befghi 72.8 ± 0.9fg 73.6 ± 1.3g 81.6 ± 1.1hi 83.1 ± 0.8i
Turbidity (NTU)
Colour L
a
b
1026 173 99 102 96 238 219 76 56
35.46 43.69 43.53 48.45 48.32 38.79 42.26 45.03 49.56
8.50 3.55 2.87 0.44 1.44 6.88 4.42 2.72 0.22
32.52 34.30 32.41 28.11 31.93 38.93 36.79 34.12 27.12
TSS (°Brix)
pH
Density ( 103 kg/m3)
Viscosity (m Pa s)
AIS (wt.%)
Sugar yield (%)
14.2 14.6 14.6 14.6 14.6 14.4 14.6 14.8 14.8
4.05 3.82 3.81 3.82 3.82 3.85 3.84 3.61 3.55
1.10 1.09 1.09 1.09 1.09 1.09 1.09 1.10 1.10
3.33 1.27 1.20 1.27 1.20 1.27 1.27 1.13 1.13
1.12 0.58 0.56 0.54 0.56 0.60 0.62 0.46 0.50
2.9 ± 0.1a 10.8 ± 0.2bfghi 11.1 ± 0.1bcfghi 10.8 ± 0.2bdfghi 11.2 ± 0.2befghi 10.5 ± 0.1fg 10.7 ± 0.2g 12.1 ± 0.2hi 12.3 ± 0.1i
PG addition – 5 U/g mash; Incubation at 50 °C for 2 h. a–i Means along a column with different superscripts are significantly different at p < 0.05 (n = 3). A NTU, Nephelometric turbidity units; AIS, Alcohol insoluble solids; SmF, Submerged fermentation; SSF, Solid-state fermentation; IMP, Integrated membrane process; AAP, Alginate affinity purification. For other abbreviations see Table 1.
process almost completely eliminated other PGs and contaminant proteins in the SmF- and SSF-cultures (Nakkeeran et al., 2009). Since apple mash processed using AAP-PGs showed more turbidity, the highly active PG of A. carbonarius would not be attractive for commercial application. IMP employing microfiltration (MF) and ultrafiltration (UF) membranes retained all the PGs. The process did not eliminate protease and amylase, since they were similar in molecular mass as that of PG (Nakkeeran et al., 2009, 2010). The presence of these enzymes in IMP processed PG preparations (Table 3) apparently further aided cell wall hydrolysis sufficient to obtain a performance comparable to those of the A. niger commercial enzymes. A reduction in viscosity of juice by about 66% was observed with the PGs after enzyme treatment of mash. Singh and Gupta (2004) reported a viscosity drop of 36% during the enzymatic clarification of commercial apple juice and Busto et al. (2006) reported a reduction of 35% during enzymatic treatment of apple pectin, but reductions in juice viscosity as high as 82–91% have been reported during enzymatic treatment of apple mash with different commercial pectolytic enzymes (Oszmianski et al., 2009).
Table 3 Enzyme activities of various PG samplesA.
A
Type of PG
PG (U/g mash)
Protease ( 103 U/g mash)
Amylase ( 102 U/g mash)
PG specific activity (U/mg protein)
SmF-Crude SSF-Crude SmF-IMP SSF-IMP SmF-AAP SSF-AAP Commercial-1 Commercial-2
5 5 5 5 5 5 5 5
0.4 2.6 0.8 5.8 ND ND 3.5 4.1
0.7 4.1 0.9 4.6 ND ND 37 44
1190 530 5590 640 9770 2450 330 120
ND, Not detected; For other abbreviations see Tables 1 and 2.
The differences in performances could be ascribed to many factors, such as type of enzyme preparation, apple variety, treatment and pressing conditions. There was no considerable difference in pH, colour and density of juice obtained from treated apple mashes with various PGs (Table 2).
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