food and bioproducts processing 9 0 ( 2 0 1 2 ) 385391

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Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Pineapple wastes: A potential source for bromelain extraction

Sunantha Ketnawa a , Phanuphong Chaiwut b , Saroat Rawdkuen a,
a b

Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, Thailand School of Cosmetic Science, Mae Fah Luang University, Muang, Chaing Rai 57100, Thailand

a b s t r a c t
This study investigates the isolation and characterization of bromelain extract from the wastes of Nang Lae and Phu Lae pineapple cultivars (economical fruits of Chiang Rai province, Thailand). The waste portions such as the peel, core, stem and crown were 2940%, 910%, 25% and 24% (w/w), respectively. The extract of crown from both cultivars gave the highest proteolytic activity and protein contents, while the extract from the stem exhibited the lowest values. SDSPAGE showed that the major protein band in the extracts was 28 kDa. Activity staining of the crown extracts from both cultivars conrmed that the major protein band showed caseinolytic activity on the casein substrate-gel. All of the crude extracts from both cultivars gave high caseinolytic activity (>80% relative) in a board pH range (39). The optimum temperatures for all crude extracts were about 5060 C. This study founded that there is much added value into local Thailand pineapple wastes because of bromelain extraction. Crown Copyright 2012 Published by Elsevier B.V. on behalf of The Institution of Chemical Engineers. All rights reserved. Keywords: Bromelain; Extraction; Nang Lae; Phu Lae; Pineapple; Waste utilization

1.

Introduction

Thailand is the biggest exporter of cannery pineapple around the world. In 2008, 2.5 million tons of pineapples were produced (FAO, 2008). Of that amount, 520,000 tons and 150,000 tons were exported as canned pineapple and pineapple juice, respectively. Chiang Rai province is one of the main areas for pineapple cultivating, especially in Nang Lae district, where pineapple is produced year-round (MOAC, 2008). In 2008, they produced around 15,00018,000 tons. During pineapple processing, the crown and stem are cut off before peeling. The core is then removed for further processing. These wastes (peel, core, stem, crown and leaves) generally account for 50% (w/w) of total pineapple weight. Therefore, with increasing pineapple production, pineapple wastes are also proportionally increasing. Waste disposal represents a growing problem since it is usually prone to microbial spoilage and it causes serious environmental problems. The utilization of waste would be an innovation to handle the great deal of waste from processing. Pineapple wastes are found to have potential uses as raw materials that can be converted into value-added products. In

agricultural, waste is occasionally utilized as a fertilizer or animal feed. The peel is a rich source of cellulose, hemicelluloses and other carbohydrates. It has been used to produce paper, banknotes, and cloth (Bartholomew et al., 2003). The core waste could be used for the production of frozen pineapple juice concentrates or extracted juice for alcoholic beverages or for vinegar (Thanong, 1985). In addition, the waste from pineapple has been used as a nutrient substance in culture broth (Nigam, 1998) and cellulose production (Omojasola et al., 2008). Moreover, the pineapple wastes have also been used as substrates for the production of methane, ethanol, citric acid and antioxidant compounds (Tanaka et al., 1999; Nigam, 1999; Chau and David, 1995; Kumar et al., 2003; Imandi et al., 2008). The utilization of pineapple wastes as a source of bioactive compounds, especially in proteolytic enzymes, is an alternative means. Bromelain and other cysteine proteases are well known enzymes present in different parts of pineapple (Ketnawa et al., 2010; Rolle, 1998; Schieber et al., 2001). Bromelain has been used commercially in the food industry, in certain cosmetics and in dietary supplements (Uhlig, 1998; Walsh, 2002). It is used for meat tenderizing, brewing, baking, as well as for the production of protein hydrolysates

Corresponding author. Tel.: +66 5393 6752; fax: +66 5393 6739. E-mail address: [email protected] (S. Rawdkuen). Received 12 March 2010; Received in revised form 11 December 2010; Accepted 21 December 2011

0960-3085/$ see front matter Crown Copyright 2012 Published by Elsevier B.V. on behalf of The Institution of Chemical Engineers. All rights reserved.

doi:10.1016/j.fbp.2011.12.006

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(Ketnawa and Rawdkuen, 2011; Walsh, 2002). Other applications are in tanning, for leather and textile industries, hair removal, wool, skin softening, and detergent formulations (Uhlig, 1998; Subhabrata and Mayura, 2006). Moreover, bromelain has been used as a folk medicine, a wound healer, an anti-inammatory, and an anti-diarrhea and digestive aid (Bitange et al., 2008; Koh et al., 2006). Because of this very wide range of applications, commercial bromelain is very expensive costing up to 2400 USD/kg. The objectives of this study were to extract bromelain from the pineapple wastes of the two cultivars, Nang Lae and Phu Lae, and to investigate some biochemical characteristics of the extracts.

and showed an absorbance of 275 nm indicated by the soluble peptides. One unit of protease activity was dened as the amount of enzyme, releasing a product equivalent to 1 g of tyrosine min1 ml1 under the standard assay conditions.

2.5.

Protein content determination

Protein present in the crude enzyme extract was measured according to the Bradford method (1976) using BSA as a standard.

2.6. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) 2.6.1. Protein pattern

2.
2.1.

Materials and methods

Chemicals

Bovine serum albumin (BSA), casein, l-tyrosine, glycine, sodium dodecyl sulfate (SDS) and Coomassie Brilliant Blue R250 were purchased from Fluka, Switzerland. Bromelain from pineapple stem, acrylamide, N,N,N ,N -methylene bisacrylamide, betamercaptoethanol (ME) were obtained from Sigma-Aldrich Co., LLC, USA. Ethylene diaminetetraacetic acid (EDTA) and trichloroacetic acid (TCA) were procured from BDH, UK. A molecular weight marker was obtained from Thermo Scientic, USA (Pierce , Cat # 26681). All other chemicals used in the experiment were analytical grade.

2.2.

Raw material

SDSPAGE was carried out by the method of Laemmli (1970) using 15% separating and 4% stacking gels. The samples were mixed with the sample buffer (0.5 M TrisHCl, pH 6.8, 0.5% bromophenol blue, 10% glycerol, and 2% SDS) with and without ME at a ratio of 1:1 for reducing and non-reducing condition, respectively. The mixture was then boiled for 3 min. Four micrograms of protein were loaded in each well and then subjected to separate at 15 mA/gel by using Mini Protean Tetra Cell units (Bio-Rad Laboratories, Inc, Richmond, CA, USA). After separation, the protein was stained with Coomassie Brilliant Blue R-250 and destained with a methanolacetic acid solution. A broad-range molecular weight standard marker containing myosin (215 kDa), phosphorylase B (120 kDa), bovine serum albumin (84 kDa), ovalbumin (60 kDa), carbonic anhydrase (39.2 kDa), trypsin inhibitor (28 kDa), and lysozyme (18.3 kDa) was used.

The pineapple (Ananus comosus L.) from Nang Lae and Phu Lae cultivars (Fig. 1A) were collected from a plantation in the Nang Lae district of Chiang Rai province, Thailand. The fruits were washed, air dried and then manually peeled. The different wastes (peel, core, crown, and stem as in Fig. 1B) were separated and then stored at 4 C for the experiments. Each waste portion was determined and reported as a percentage of the proportion of a pineapple.

2.6.2.

Activity staining

2.3.

Preparation of crude extract

Each pineapple waste was chopped into small pieces before being blended (Philips HR-2011 Blender, China) with cold distilled water at a 1:1 ratio for 3 min. The resulting blend was ltered through a cheese cloth and then centrifuged at 10,000 g at 4 C for 20 min. The obtained supernatant (crude enzyme extract) was collected, recorded and used for pH measurement by using a pH meter (Eutech Instruments pH 510, Singapore). The total soluble solids were also measured by using a hand refractometer (Atago N1-E, Japan) and it was reported as degrees Brix.

The bromelain activity in the protein band separated on the SDSPAGE was veried by using activity staining according to the method of Garcia Carreno et al. (1993) with slight modication. Each well was loaded with 2 g of the protein. After electrophoresis, the gel was immersed in 50 ml of 2% (w/v) casein in 0.05 M sodium phosphate buffer (pH 7.0), containing 0.03 M cysteine and 0.006 M EDTA, with a constant agitation at 4 C for 45 min. The gel was then incubated at 37 C for 30 min and then rinsed with distilled water, xed, stained and destained as mentioned above. The bromelain activity was observed by developing clear zones against a dark background. The apparent molecular weight (MW) of the bromelain was estimated by comparing the reference distance (Rf) with those of molecular weight standard protein markers.

2.7.

pH prole assay

2.4.

Proteolytic activity determination

The proteolytic activity of the crude enzyme extracts was determined by the Murachi method (1976), using casein and l-tyrosine as a substrate and a standard, respectively. The extract (1.0 ml) was mixed with 1.0 ml of a reaction cocktail (contained 1% (w/v) of casein, 0.03 M cysteine, 0.006 M EDTA in 0.05 M phosphate, and a buffer pH 7.0). The reaction was carried out at 37 C and was stopped by the addition of 3 ml of 5% (w/v) TCA. The reaction mixture was then centrifuged at 8000 g for 10 min. The obtained supernatant was measured

The pH prole was determined by assaying the proteolytic activity in different pHs (310). Glycine (pH 3), sodium acetate (pH 45), sodium phosphate buffer (pH 67), TrisHCl buffer (pH 810) were used. The residual proteolytic activity was measured and expressed as the relative proteolytic activity.

2.8.

Temperature prole assay

Proteolytic activity of crude extract was performed at different temperatures (40, 50, 60, 70, 80, 90, and 100 C) for 10 min. The assay was measured as mentioned above by using casein as a substrate. The caseinolytic activity was expressed as the relative proteolytic activity, compared with that of the control.

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Fig. 1 Morphology of Nang Lae and Phu Lae pineapple fruits (A) and their wastes (B).

2.9.

Statistical analysis

Analysis of variance (ANOVA) was used to analyze the data from triplicate measurements. Differences between means were evaluated by Duncans multiple range test by using the SPSS (Version 11.5).

3.
3.1.

Results and discussion

Proportion of pineapples wastes

The Nang Lae and Phu Lae fruits were divided into different parts as presented in Table 1. Of the pineapple wastes, the peels were the largest portion (3042%, w/w), especially the peels of the Phu Lae pineapple (42%, w/w). Other proportions including the cores, stems and crowns were 910, 25 and 24% (w/w), respectively. In addition, the Nang Lae fruits had around 50% (w/w) esh while that of Phu Lae was about 42% (w/w). As a result, the pineapple wastes (peel, core stem and crown) accounted for 50% (w/w) of total pineapple weight. Due to increasing production, approximately 2.8 million tons of the peels and 370,000 tons of the crowns are generated annually (FAO, 2008). Nang Lae pineapples have more edible portions than Phu Lae because they are bigger in size.

3.2.

Some characteristics of crude extracts

Crude extracts were prepared by extracting the pineapple wastes (100 g) with distilled water at a ratio of 1:1 (w/v) and then the pH, total soluble solid, protein content and enzyme activity were all measured. The results are presented

in Table 2. The pH of the crude extracts from Nang Lae was the same as Phu Lae at around 4.05.0. The pH of the crown portion showed the highest value (4.85.19) while the peels of both cultivars gave the lowest value (4.00). The main organic acids of ripe pineapple fruit are citric and malic acid (Bartolome et al., 1995). The low pH value indicated the high acidity of citric and malic acid in the extracts. In both cultivars, the pH value was close to the pH of a Smooth Cayenne cultivar, which was 3.54 (Bartolome et al., 1995). The total soluble solid of Nang Lae and Phu Lae crude extracts were 3.04.7 and 2.66.3 Brix, respectively. Bartolome et al. (1995) reported a total soluble solid of Smooth Cayenne of 12.48 Brix, and the total soluble sugars detected in the cultivar were sucrose, fructose and glucose in the amounts of 4.50, 2.21, and 1.45 g/l, respectively. The distinct total soluble solids might be due to the differences of cultivars and plantation area. Bartholomew et al. (2003) reported that the cultivar and cultivation affect the pH and total soluble solids. According to 100 g of pineapple waste materials, the extract from the crown of both cultivars exhibited the highest total protein contents (141 and 220 mg for Phu Lae and Nang Lae, respectively) and total proteolytic activity (322,000 and 173,000 units for Phu Lae and Nang Lae, respectively). The lowest protein content was found in the core extract of the Phu Lae fruit (27 mg) and the stem extract of the Nang Lae fruit (30 mg). Protein content was much lower in the stem and the core portion compared with the other wastes (p < 0.05). Same with the protein content, the highest total protease activity was found in the crown portion of both cultivars, and the lowest activity was found in the stem. To obtain the bromelain, the pineapple peel provided the second major part behind the crown. The

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Table 1 Proportion of pineapple fruits Nang Lae and Phu Lae cultivars. Cultivar proportion Weight (g)
Peel Core Stem Crown Flesh Total
a

Nang Lae % (w/w)

a

Phu Lae Weight (g)

159.86 40.60 9.26 10.20 158.94 378.86 2.64a 3.38b 3.67c 0.98c 1.04a 11.32

% (w/w)
42.20 10.72 2.44 2.69 41.95 100.00 3.51a 1.46b 0.45c 0.15c 1.02a 5.53

143.40 44.61 26.55 22.48 239.86 476.54

8.53 b 0.69c** 4.05d 1.62d 0.10a 8.26

30.09 9.36 5.57 4.72 50.33 100.00

3.96b 0.76c 1.20c 0.38c 4.20a 8.22

Means SD from triplicate determinations. Different letters in the same column indicate the signicant differences (p < 0.05).

differences in enzyme activity and protein content in each portion are probably due to the different types of enzymes consisted in the pineapple, such as enzymes from the stem, and from the fruit: ananain and comosain (Maurer, 2001; Hale et al., 2005).

3.3. 3.3.1.

Electrophoresis analysis Protein patterns

Protein patterns of the crude extracts from Nang Lae and Phu Lae pineapple wastes under non-reducing and reducing conditions are shown in Fig. 2A. The results show that the protein components in the crude extract were almost the same for both cultivars. From the protein patterns under non-reducing condition, the main protein components in the waste extracts showed MW of 39.2, 28, and 18.3 kDa. The small proteins had MW below 18.3 kDa. Stem bromelain (lane 9) was used as a reference to show the MW of 28 kDa. High protein band intensity was found in the crown portion with an MW of 28 kDa. Interestingly, their protein band (MW of 28 kDa) was the major component in the crown extract of both cultivars (lanes 5 and 6). Umesh et al. (2008) reported that the bromelain extracted from pineapple cores was found to be around 26 kDa by using SDSPAGE analysis. Maurer (2001) reported that the bromelain extracted from stems and fruits were 23.8 kDa and 23 kDa, respectively.

crude extract from the peels or the stems of both cultivars. The Phu Lae pineapple cores in non-reducing conditions showed no clear zone in the crude extracts. In contrast, the crude extract from the core of Nang Lae pineapple and the crowns of both cultivars showed the clear zone at MW 39.2 kDa. The presence of this clear band in activity staining of non-reducing condition suggested that proteases content in those extracts, especially in the crown, were higher than those of other portions. There were some protein bands that did not show caseinolytic activity staining (Fig. 2B). This can be explained by the presence of other non-proteases in the pineapple extracts. There are consistent reports of the presence of peroxidase, acid phosphatase, and several protease inhibitors resulting in no protease activity (Bitange et al., 2008; Umesh et al., 2008). There was no clear zone observation of all crude extracts in activity staining under the reducing condition (Fig. 2B). This result indicated that the bromelain is stabilized by the disulde bond. The presence of reducing agents broke of this bond and enzyme occurred denaturation.

3.4.

pH prole

3.3.2.

Activity staining

To verify the band of bromelain, activity staining was performed by substrate (casein) gel electrophoresis. Fig. 2B shows the activity staining of the crude extracts from Nang Lae and Phu Lae pineapple wastes. No clear zone was observed in the

The effect of pH on proteolytic activity of the crude extract from both Nang Lae and Phu Lae pineapple was measured and reported as a relative protease activity against the control. The pH ranges of 310 were performed in each crude extract (Fig. 3). All crude fractions from the two cultivars exhibited a board pH activity prole. The crude extracts from both cultivars produced high caseinolytic activity within a pH range of 6.58.0, while maximum activity was found at around pH 7.0. The crude extract from the peels, cores, and crowns of Nang Lae pineapple showed the highest caseinolytic activity at pH

Table 2 Characteristics of crude extract from pineapple wastes. Proportion

Nang Lae Peel Core Stem Crown Phu Lae Peel Core Stem Crown
a b

pH
4.02 4.27 4.76 5.19 4.01 4.09 4.64 4.80 0.30a b 0.24b** 0.16b 0.26a 0.13b 0.22b 0.22a 0.13a

TSS ( Brix)
4.33 4.73 3.00 3.00 4.43 6.27 2.63 3.43 0.58ab 0.87a 1.00b 0.00b 0.81b 0.64a 0.15c 0.40bc

Volume of extract (ml)b

163.5 175.5 140.5 133.5 154.5 164.0 149.9 113.2 0.71b 0.54a 0.61c 0.44d 3.54b 0.66a 0.25c 0.47d

Total protein (mg)

132.4 45.4 29.8 220.5 70.7 27.1 28.1 141.0 1.40b 0.87c 0.76d 3.65a 0.46b 1.83c 1.85c 3.30a

Total activity (unit)

90,653 36,111 14,435 172,964 118,920 42,482 17,068 322,734 1.08b 1.62c 2.26d 1.29a 2.95b 2.22c 0.44d 1.67a

Means SD from triplicate determinations. Each portion was used 100 g for each extraction. Different letters in the same column indicate the signicant differences (p < 0.05).

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Fig. 2 Protein patterns (A) and activity staining (B) of enzyme extracts from Nang Lae and Phu Lae pineapple under non-reducing and reducing conditions. Nang Lae (lanes 1, 3, 5 and 7) and Phu Lae cultivars (lanes 2, 4, 6 and 8) pineapple. M: molecular weight marker (kDa); lanes 1 and 2: peel; lanes 3 and 4: core; lanes 5 and 6: crown; lanes 7 and 8: stem; lane 9: commercial stem bromelain. 7.0, while those of Phu Lae pineapple exposed the highest caseinolytic activity at pH 8, 7, 6, respectively. Nonetheless, the crude extract from the stem of both cultivars possessed the highest caseinolytic activity at pH 8.0. This is probably due to the different types of enzymes in the stem crude extract from those of the others. Proteolytic enzymes from the ripe fruit of Bromelia antiacantha Bertol (Bromeliaceae) exhibited high caseinolytic activity (higher than 80%) in a broad pH range (59), with two maximum pH level at 6.0 and at 9.0 (Valles et al., 2007). Koh et al. (2006) reported that the enzyme activity of pineapple produced in jeju-island, South Korea was found to be optimal at pH 7. The activity of bromelain dramatically decreased at acidic conditions of 34 and also at alkaline conditions of 910. In this regard, the isolated enzyme is unique, and therefore it might be useful for application in foods and in the pharmaceutical industry.

3.5.

Thermal prole

For the thermal prole, the crude extract of both Nang Lae and Phu Lae pineapple was determined at a temperature ranging from 30 to 90 C. The relative proteolytic activity against casein was calculated and presented in Fig. 4. The highest activity was found at 50 C for all crude extracts from Nang Lae and 60 C for Phu Lae cultivars. As the incubation temperature used was increased, the relative proteolytic activity constantly decreased and reached the lowest point at 90 C. This result is similar to previous reports. The optimum temperature was observed at 63 C of the proteolytic enzymes from ripe fruits of B. antiacantha Bertol (Bromeliaceae) (Valles et al., 2007). Koh et al. (2006) also reported that the enzyme activity of pineapple produced in jeju-island was found to be optimal at the temperature of 60 C. Pineapple obtained from Imphal, Manipur, India

Peel

Core

Stem

Crown

Peel

Core

Stem

Crown

Relative proteolytic activity (%)

Relative proteolytic activity (%)

105 100 95 90 85 80 75 70 2 3 4 5 6 7 8

Nang Lae

105 100 95 90 85 80 75 70 2 3 4 5 6 7 8

Phu Lae

10

11

10

11

pH

pH

Fig. 3 Effect of pH on caseinolytic activity of crude extract from peel, core, stem, and crown of Nang Lae and Phu Lae pineapples.

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Peel 100 Core

food and bioproducts processing 9 0 ( 2 0 1 2 ) 385391

Stem

Crown

Peel 100

Core

Stem

Crown

Nang Lae
Relative proteolytic activity (%)

Relative proteolytic activity (%)

90 80 70 60 50 40 30 20 10 0

Phu Lae

90 80 70 60 50 40 30 20 10 0

20

30

40

50

60

70

80

90

100

20

30

40

50

60

70

80

90

100

Temperature ('C)

Temperature ('C)

Fig. 4 Effect of temperature on caseinolytic activity of crude extract from peel, core, stem, and crown of Nang Lae and Phu Lae pineapples. showed the optimum temperature at 60 C (Subhabrata and Mayura, 2006). As the temperature increases, more molecules have enough kinetic energy to undergo the reaction. If the temperature is raised above the optimum point, the kinetic energy of the enzyme and water molecules is so great that the structure of the enzyme molecule starts to be disrupted (Switzer and Garrity, 1999). Therefore, a decrease in activity was detected. The knowledge of optimum temperature is of use to explore the usefulness of the enzyme either directly or after modications. As reported, bromelain is remarkably heat stable, retaining proteolytic activity between 40 and 60 C where most enzymes are destroyed or denatured.
Bartolome, A.P., Rupbrez, P., Carmen, F., 1995. Pineapple fruit: morphological characteristics, chemical composition and sensory analysis of Red Spanish and Smooth Cayenne cultivars. Food Chem. 53, 7579. Bitange, N.T., Zhang, W., Shi, Y.X., Wenbin, Z., 2008. Therapeutic application of pineapple protease (bromelain). Pakistan J. Nutr. 7, 513520. Bradford, M.M., 1976. A rapid and sensitive method for the quantication of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72, 248254. Chau, T., David, A.M., 1995. Pineapple wastea novel substrate for citric acid production by solid state fermentation. Biotechnol. Lett. 17, 11071110. FAO, 2008. Statistical Yearbook. Food and Agriculture Organization of the United Nations, USA, Available at: www.fao.org (accessed 01.07.09). Garcia Carreno, F.C., Dimes, C.E., Haard, N.F., 1993. Substrate gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors. Anal. Biochem. 214, 6569. Hale, L.P., Greer, P.K., Trinh, C.T., James, C.L., 2005. Proteinase activity and stability of natural bromelain preparations. Int. Immunol. 5, 783793. Imandi, S.B., Bandaru, V.V., Somalanka, S.R., Bandaru, S.R., Garapati, H.R., 2008. Application of statistical experimental designs for the optimization of medium constituents for the production of citric acid from pineapple waste. Bioresour. Technol. 99, 44454450. Ketnawa, S., Rawdkuen, S., 2011. Application of bromelain extract for muscle foods tenderization. Food Nutri. Sci. 2, 393401. Ketnawa, S., Rawdkuen, S., Chaiwut, P., 2010. Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar. Biochem. Eng. J. 52, 205211. Koh, J., Kang, S.M., Kim, S.J., Cha, M.K., Kwon, Y.J., 2006. Effect of pineapple protease on the characteristics of protein bers. Fiber Polym. 7, 180185. Kumar, D., Jain, V.K., Shanker, G., Srivastava, A., 2003. Utilisation of fruits waste for citric acid production by solid state fermentation. Process Biochem. 38, 17251729. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227, 680685. Murachi, T., 1976. Bromelain enzymes. In: Lorand, L. (Ed.), Methods in Enzymology, vol. 19. Academic Press, New York, pp. 475485. Maurer, H.R., 2001. Bromelain: biochemistry, pharmacology and medical use. Cell. Mol. Life Sci. 9, 12341245. MOAC, Ofce of Agricultural Economics, Ministry of Agriculture and Cooperatives, 2008. Statistical Yearbook, Thailand, Available at: www.moac.go.th (accessed 01.07.09). Nigam, J.N., 1998. Single cell protein from pineapple canner efuent. World J. Microbiol. Biotechnol. 14, 693696.

4.

Conclusion

For both Nang Lae and Phu Lae pineapple, the peel is the largest waste portion followed by the core, stem, and crown. The crown extract of both cultivars contained bromelain as a major enzyme with the MW 28 kDa. Most of the extracts exhibited the highest caseinolytic activity at a pH of around 7.0, whereas those of stem extracts were 8.0. All extracts of Nang Lae pineapple produced the highest activity at 50 C. For Phu Lae extracts, the highest activity occurred at 60 C. The peel seems to have the most promise for bromelain extraction because it accounts for the largest waste proportion. The pineapple waste residues after bromelain extraction contain high amount of ber. It would be useful to suggest further application of these residues for paper and paper board productions.

Acknowledgements
The authors would like to thank Mae Fah Luang University and National Research Council of Thailand (NRCT) for nancial support under the project No. PK/2553-40. We also thank Prof. Dr. Soottawat Benjakul, Department of Food Technology, Prince of Songkla University for his assistance in technical writing and Prof. Matthew Robert Ferguson, Language Center, Naresuan University for kindly providing corrections for the manuscript.

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