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food and bioproducts processing 9 0 ( 2 0 1 2 ) 385–391

Contents lists available at SciVerse ScienceDirect

Food and Bioproducts Processing

j our nal ho mepage: www.elsev ier .com/ locate / fbp

ineapple wastes: A potential source for bromelainxtraction

unantha Ketnawaa, Phanuphong Chaiwutb, Saroat Rawdkuena,∗

Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Muang, Chiang Rai 57100, ThailandSchool 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 29–40%, 9–10%, 2–5% and 2–4% (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. SDS–PAGE showed that the major protein band in the extracts was ∼28 kDa. Activity staining of the

crown extracts from both cultivars confirmed 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 (3–9). The optimum temperatures for all crude extracts were about 50–60 ◦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

1998; Walsh, 2002). It is used for meat tenderizing, brewing,

. Introduction

hailand is the biggest exporter of cannery pineapple aroundhe world. In 2008, ∼2.5 million tons of pineapples wereroduced (FAO, 2008). Of that amount, 520,000 tons and50,000 tons were exported as canned pineapple and pineap-le juice, respectively. Chiang Rai province is one of the mainreas for pineapple cultivating, especially in Nang Lae dis-rict, where pineapple is produced year-round (MOAC, 2008).n 2008, they produced around 15,000–18,000 tons. Duringineapple processing, the crown and stem are cut off beforeeeling. The core is then removed for further processing.hese wastes (peel, core, stem, crown and leaves) generallyccount for 50% (w/w) of total pineapple weight. Therefore,ith increasing pineapple production, pineapple wastes are

lso proportionally increasing. Waste disposal represents arowing problem since it is usually prone to microbial spoilagend it causes serious environmental problems. The utilizationf waste would be an innovation to handle the great deal ofaste from processing.

Pineapple wastes are found to have potential uses as raw

aterials that can be converted into value-added products. In

∗ Corresponding author. Tel.: +66 5393 6752; fax: +66 5393 6739.E-mail address: saroat@mfu.ac.th (S. Rawdkuen).Received 12 March 2010; Received in revised form 11 December 2010;

960-3085/$ – see front matter Crown Copyright © 2012 Published by Elsevier B.oi:10.1016/j.fbp.2011.12.006

agricultural, waste is occasionally utilized as a fertilizer or ani-mal feed. The peel is a rich source of cellulose, hemicellulosesand other carbohydrates. It has been used to produce paper,banknotes, and cloth (Bartholomew et al., 2003). The corewaste could be used for the production of frozen pineapplejuice concentrates or extracted juice for alcoholic beveragesor for vinegar (Thanong, 1985). In addition, the waste frompineapple has been used as a nutrient substance in culturebroth (Nigam, 1998) and cellulose production (Omojasola et al.,2008). Moreover, the pineapple wastes have also been used assubstrates for the production of methane, ethanol, citric acidand 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 bioactivecompounds, especially in proteolytic enzymes, is an alter-native means. Bromelain and other cysteine proteases arewell known enzymes present in different parts of pineap-ple (Ketnawa et al., 2010; Rolle, 1998; Schieber et al., 2001).Bromelain has been used commercially in the food indus-try, in certain cosmetics and in dietary supplements (Uhlig,

Accepted 21 December 2011

baking, as well as for the production of protein hydrolysates

V. on behalf of The Institution of Chemical Engineers. All rights reserved.

386 food and bioproducts processing 9 0 ( 2 0 1 2 ) 385–391

(Ketnawa and Rawdkuen, 2011; Walsh, 2002). Other applica-tions are in tanning, for leather and textile industries, hairremoval, wool, skin softening, and detergent formulations(Uhlig, 1998; Subhabrata and Mayura, 2006). Moreover, brome-lain has been used as a folk medicine, a wound healer, ananti-inflammatory, and an anti-diarrhea and digestive aid(Bitange et al., 2008; Koh et al., 2006).

Because of this very wide range of applications, commer-cial bromelain is very expensive costing up to 2400 USD/kg.The objectives of this study were to extract bromelain fromthe pineapple wastes of the two cultivars, Nang Lae and PhuLae, and to investigate some biochemical characteristics of theextracts.

2. Materials and methods

2.1. Chemicals

Bovine serum albumin (BSA), casein, l-tyrosine, glycine,sodium dodecyl sulfate (SDS) and Coomassie Brilliant Blue R-250 were purchased from Fluka, Switzerland. Bromelain frompineapple stem, acrylamide, N,N,N′,N′-methylene bisacry-lamide, betamercaptoethanol (�ME) were obtained fromSigma-Aldrich Co., LLC, USA. Ethylene diaminetetraacetic acid(EDTA) and trichloroacetic acid (TCA) were procured from BDH,UK. A molecular weight marker was obtained from ThermoScientific, USA (Pierce®, Cat # 26681). All other chemicals usedin the experiment were analytical grade.

2.2. Raw material

The pineapple (Ananus comosus L.) from Nang Lae and Phu Laecultivars (Fig. 1A) were collected from a plantation in the NangLae district of Chiang Rai province, Thailand. The fruits werewashed, air dried and then manually peeled. The differentwastes (peel, core, crown, and stem as in Fig. 1B) were sepa-rated and then stored at 4 ◦C for the experiments. Each wasteportion was determined and reported as a percentage of theproportion of a pineapple.

2.3. Preparation of crude extract

Each pineapple waste was chopped into small pieces beforebeing blended (Philips HR-2011 Blender, China) with colddistilled water at a 1:1 ratio for 3 min. The resulting blendwas filtered through a cheese cloth and then centrifuged at10,000 × g at 4 ◦C for 20 min. The obtained supernatant (crudeenzyme extract) was collected, recorded and used for pH mea-surement by using a pH meter (Eutech Instruments pH 510,Singapore). The total soluble solids were also measured byusing a hand refractometer (Atago N1-E, Japan) and it wasreported as degrees Brix.

2.4. Proteolytic activity determination

The proteolytic activity of the crude enzyme extracts wasdetermined by the Murachi method (1976), using casein andl-tyrosine as a substrate and a standard, respectively. Theextract (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 EDTAin 0.05 M phosphate, and a buffer pH 7.0). The reaction wascarried out at 37 ◦C and was stopped by the addition of 3 ml of

5% (w/v) TCA. The reaction mixture was then centrifuged at8000 × g for 10 min. The obtained supernatant was measured

and showed an absorbance of 275 nm indicated by the solu-ble peptides. One unit of protease activity was defined as theamount of enzyme, releasing a product equivalent to 1 �g oftyrosine min−1 ml−1 under the standard assay conditions.

2.5. Protein content determination

Protein present in the crude enzyme extract was measuredaccording to the Bradford method (1976) using BSA as a stan-dard.

2.6. Sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS–PAGE)

2.6.1. Protein patternSDS–PAGE was carried out by the method of Laemmli (1970)using 15% separating and 4% stacking gels. The sampleswere mixed with the sample buffer (0.5 M Tris–HCl, pH6.8, 0.5% bromophenol blue, 10% glycerol, and 2% SDS)with and without �ME at a ratio of 1:1 for reducing andnon-reducing condition, respectively. The mixture was thenboiled for 3 min. Four micrograms of protein were loadedin each well and then subjected to separate at 15 mA/gelby using Mini Protean Tetra Cell units (Bio-Rad Laboratories,Inc, Richmond, CA, USA). After separation, the protein wasstained with Coomassie Brilliant Blue R-250 and destainedwith a methanol–acetic acid solution. A broad-range molec-ular weight standard marker containing myosin (215 kDa),phosphorylase B (120 kDa), bovine serum albumin (84 kDa),ovalbumin (60 kDa), carbonic anhydrase (39.2 kDa), trypsininhibitor (28 kDa), and lysozyme (18.3 kDa) was used.

2.6.2. Activity stainingThe bromelain activity in the protein band separated on theSDS–PAGE was verified by using activity staining according tothe method of Garcia Carreno et al. (1993) with slight modi-fication. Each well was loaded with 2 �g of the protein. Afterelectrophoresis, the gel was immersed in 50 ml of 2% (w/v)casein in 0.05 M sodium phosphate buffer (pH 7.0), containing0.03 M cysteine and 0.006 M EDTA, with a constant agitationat 4 ◦C for 45 min. The gel was then incubated at 37 ◦C for30 min and then rinsed with distilled water, fixed, stained anddestained as mentioned above. The bromelain activity wasobserved by developing clear zones against a dark background.The apparent molecular weight (MW) of the bromelain wasestimated by comparing the reference distance (Rf) with thoseof molecular weight standard protein markers.

2.7. pH profile assay

The pH profile was determined by assaying the proteolyticactivity in different pHs (3–10). Glycine (pH 3), sodium acetate(pH 4–5), sodium phosphate buffer (pH 6–7), Tris–HCl buffer(pH 8–10) were used. The residual proteolytic activity was mea-sured and expressed as the relative proteolytic activity.

2.8. Temperature profile assay

Proteolytic activity of crude extract was performed at differ-ent 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 therelative proteolytic activity, compared with that of the control.

food and bioproducts processing 9 0 ( 2 0 1 2 ) 385–391 387

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

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.9. Statistical analysis

nalysis of variance (ANOVA) was used to analyze the datarom triplicate measurements. Differences between meansere evaluated by Duncan’s multiple range test by using thePSS (Version 11.5).

. Results and discussion

.1. Proportion of pineapples wastes

he Nang Lae and Phu Lae fruits were divided into differentarts as presented in Table 1. Of the pineapple wastes, theeels were the largest portion (30–42%, w/w), especially theeels of the Phu Lae pineapple (42%, w/w). Other proportions

ncluding the cores, stems and crowns were 9–10, 2–5 and–4% (w/w), respectively. In addition, the Nang Lae fruits hadround 50% (w/w) flesh while that of Phu Lae was about 42%w/w). As a result, the pineapple wastes (peel, core stem andrown) accounted for 50% (w/w) of total pineapple weight. Dueo increasing production, approximately 2.8 million tons of theeels and 370,000 tons of the crowns are generated annually

FAO, 2008). Nang Lae pineapples have more edible portionshan Phu Lae because they are bigger in size.

.2. Some characteristics of crude extracts

rude extracts were prepared by extracting the pineappleastes (100 g) with distilled water at a ratio of 1:1 (w/v)

nd then the pH, total soluble solid, protein content andnzyme activity were all measured. The results are presented

in Table 2. The pH of the crude extracts from Nang Lae was thesame as Phu Lae at around 4.0–5.0. The pH of the crown portionshowed the highest value (4.8–5.19) while the peels of both cul-tivars gave the lowest value (4.00). The main organic acids ofripe pineapple fruit are citric and malic acid (Bartolome et al.,1995). The low pH value indicated the high acidity of citric andmalic acid in the extracts. In both cultivars, the pH value wasclose 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 crudeextracts were 3.0–4.7 and 2.6–6.3

◦Brix, respectively. Bartolome

et al. (1995) reported a total soluble solid of Smooth Cayenne of12.48

◦Brix, and the total soluble sugars detected in the culti-

var were sucrose, fructose and glucose in the amounts of 4.50,2.21, and 1.45 g/l, respectively. The distinct total soluble solidsmight be due to the differences of cultivars and plantationarea. Bartholomew et al. (2003) reported that the cultivar andcultivation affect the pH and total soluble solids.

According to 100 g of pineapple waste materials, the extractfrom the crown of both cultivars exhibited the highest totalprotein contents (141 and 220 mg for Phu Lae and Nang Lae,respectively) and total proteolytic activity (322,000 and 173,000units for Phu Lae and Nang Lae, respectively). The lowest pro-tein 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). Pro-tein content was much lower in the stem and the core portioncompared with the other wastes (p < 0.05). Same with the pro-tein content, the highest total protease activity was found inthe crown portion of both cultivars, and the lowest activity

was found in the stem. To obtain the bromelain, the pineap-ple peel provided the second major part behind the crown. The

388 food and bioproducts processing 9 0 ( 2 0 1 2 ) 385–391

Table 1 – Proportion of pineapple fruits Nang Lae and Phu Lae cultivars.

Cultivar proportion Nang Lae Phu Lae

Weight (g) % (w/w) Weight (g) % (w/w)

Peel 143.40 ± 8.53ab 30.09 ± 3.96b 159.86 ± 2.64a 42.20 ± 3.51aCore 44.61 ± 0.69c** 9.36 ± 0.76c 40.60 ± 3.38b 10.72 ± 1.46bStem 26.55 ± 4.05d 5.57 ± 1.20c 9.26 ± 3.67c 2.44 ± 0.45cCrown 22.48 ± 1.62d 4.72 ± 0.38c 10.20 ± 0.98c 2.69 ± 0.15cFlesh 239.86 ± 0.10a 50.33 ± 4.20a 158.94 ± 1.04a 41.95 ± 1.02aTotal 476.54 ± 8.26 100.00 ± 8.22 378.86 ± 11.32 100.00 ± 5.53

a Means ± SD from triplicate determinations.∗∗ Different letters in the same column indicate the significant differences (p < 0.05).

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

3.3. Electrophoresis analysis

3.3.1. Protein patternsProtein patterns of the crude extracts from Nang Lae and PhuLae pineapple wastes under non-reducing and reducing con-ditions are shown in Fig. 2A. The results show that the proteincomponents in the crude extract were almost the same forboth cultivars. From the protein patterns under non-reducingcondition, the main protein components in the waste extractsshowed MW of 39.2, 28, and 18.3 kDa. The small proteins hadMW below 18.3 kDa. Stem bromelain (lane 9) was used asa reference to show the MW of ∼28 kDa. High protein bandintensity was found in the crown portion with an MW of28 kDa. Interestingly, their protein band (MW of 28 kDa) wasthe major component in the crown extract of both cultivars(lanes 5 and 6). Umesh et al. (2008) reported that the bromelainextracted from pineapple cores was found to be around 26 kDaby using SDS–PAGE analysis. Maurer (2001) reported that thebromelain extracted from stems and fruits were 23.8 kDa and23 kDa, respectively.

3.3.2. Activity stainingTo verify the band of bromelain, activity staining was per-formed by substrate (casein) gel electrophoresis. Fig. 2B shows

the activity staining of the crude extracts from Nang Lae andPhu Lae pineapple wastes. No clear zone was observed in the

Table 2 – Characteristics of crude extract from pineapple waste

Proportion pH TSS (◦Brix) Volume of ex

Nang LaePeel 4.02 ± 0.30ab 4.33 ± 0.58ab 163.5 ± 0.71bCore 4.27 ± 0.24b** 4.73 ± 0.87a 175.5 ± 0.54aStem 4.76 ± 0.16b 3.00 ± 1.00b 140.5 ± 0.61cCrown 5.19 ± 0.26a 3.00 ± 0.00b 133.5 ± 0.44dPhu LaePeel 4.01 ± 0.13b 4.43 ± 0.81b 154.5 ± 3.54bCore 4.09 ± 0.22b 6.27 ± 0.64a 164.0 ± 0.66aStem 4.64 ± 0.22a 2.63 ± 0.15c 149.9 ± 0.25cCrown 4.80 ± 0.13a 3.43 ± 0.40bc 113.2 ± 0.47d

a Means ± SD from triplicate determinations.b Each portion was used 100 g for each extraction.

∗∗ Different letters in the same column indicate the significant differences

crude extract from the peels or the stems of both cultivars. ThePhu Lae pineapple cores in non-reducing conditions showed noclear zone in the crude extracts. In contrast, the crude extractfrom the core of Nang Lae pineapple and the crowns of bothcultivars showed the clear zone at MW 39.2 kDa. The pres-ence of this clear band in activity staining of non-reducingcondition suggested that proteases content in those extracts,especially in the crown, were higher than those of other por-tions. There were some protein bands that did not showcaseinolytic activity staining (Fig. 2B). This can be explained bythe 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 inno protease activity (Bitange et al., 2008; Umesh et al., 2008).

There was no clear zone observation of all crude extractsin activity staining under the reducing condition (Fig. 2B). Thisresult indicated that the bromelain is stabilized by the disul-fide bond. The presence of reducing agents broke of this bondand enzyme occurred denaturation.

3.4. pH profile

The effect of pH on proteolytic activity of the crude extractfrom both Nang Lae and Phu Lae pineapple was measured andreported as a relative protease activity against the control.The pH ranges of 3–10 were performed in each crude extract(Fig. 3). All crude fractions from the two cultivars exhibited aboard pH activity profile. The crude extracts from both culti-vars produced high caseinolytic activity within a pH range of6.5–8.0, while maximum activity was found at around pH 7.0.

The crude extract from the peels, cores, and crowns of NangLae pineapple showed the highest caseinolytic activity at pH

s.

tract (ml)b Total protein (mg) Total activity (unit)

132.4 ± 1.40b 90,653 ± 1.08b 45.4 ± 0.87c 36,111 ± 1.62c

29.8 ± 0.76d 14,435 ± 2.26d 220.5 ± 3.65a 172,964 ± 1.29a

70.7 ± 0.46b 118,920 ± 2.95b 27.1 ± 1.83c 42,482 ± 2.22c

28.1 ± 1.85c 17,068 ± 0.44d 141.0 ± 3.30a 322,734 ± 1.67a

(p < 0.05).

food and bioproducts processing 9 0 ( 2 0 1 2 ) 385–391 389

Fig. 2 – Protein patterns (A) and activity staining (B) of enzyme extracts from Nang Lae and Phu Lae pineapple undernon-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:c

7cttdetebatSo3if

produced in jeju-island was found to be optimal at the temper-ature of 60 ◦C. Pineapple obtained from Imphal, Manipur, India

Fp

ommercial stem bromelain.

.0, while those of Phu Lae pineapple exposed the highestaseinolytic activity at pH 8, 7, 6, respectively. Nonetheless,he crude extract from the stem of both cultivars possessedhe highest caseinolytic activity at pH 8.0. This is probablyue to the different types of enzymes in the stem crudextract from those of the others. Proteolytic enzymes fromhe ripe fruit of Bromelia antiacantha Bertol (Bromeliaceae)xhibited high caseinolytic activity (higher than 80%) in aroad pH range (5–9), with two maximum pH level at 6.0nd at 9.0 (Valles et al., 2007). Koh et al. (2006) reported thathe enzyme activity of pineapple produced in jeju-island,outh Korea was found to be optimal at pH 7. The activityf bromelain dramatically decreased at acidic conditions of–4 and also at alkaline conditions of 9–10. In this regard, thesolated enzyme is unique, and therefore it might be usefulor application in foods and in the pharmaceutical industry.

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ineapples.

3.5. Thermal profile

For the thermal profile, the crude extract of both Nang Laeand Phu Lae pineapple was determined at a temperature rang-ing from 30 to 90 ◦C. The relative proteolytic activity againstcasein was calculated and presented in Fig. 4. The highestactivity was found at 50 ◦C for all crude extracts from Nang Laeand 60 ◦C for Phu Lae cultivars. As the incubation temperatureused was increased, the relative proteolytic activity constantlydecreased and reached the lowest point at 90 ◦C. This resultis similar to previous reports. The optimum temperature wasobserved at 63 ◦C of the proteolytic enzymes from ripe fruitsof B. antiacantha Bertol (Bromeliaceae) (Valles et al., 2007). Kohet al. (2006) also reported that the enzyme activity of pineapple

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peel, core, stem, and crown of Nang Lae and Phu Lae

390 food and bioproducts processing 9 0 ( 2 0 1 2 ) 385–391

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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 andMayura, 2006). As the temperature increases, more moleculeshave enough kinetic energy to undergo the reaction. If thetemperature is raised above the optimum point, the kineticenergy of the enzyme and water molecules is so great thatthe structure of the enzyme molecule starts to be disrupted(Switzer and Garrity, 1999). Therefore, a decrease in activitywas detected. The knowledge of optimum temperature is ofuse to explore the usefulness of the enzyme either directlyor after modifications. As reported, bromelain is remarkablyheat stable, retaining proteolytic activity between 40 and 60 ◦Cwhere most enzymes are destroyed or denatured.

4. Conclusion

For both Nang Lae and Phu Lae pineapple, the peel is thelargest waste portion followed by the core, stem, and crown.The crown extract of both cultivars contained bromelain asa major enzyme with the MW ∼28 kDa. Most of the extractsexhibited the highest caseinolytic activity at a pH of around7.0, whereas those of stem extracts were 8.0. All extracts ofNang Lae pineapple produced the highest activity at 50 ◦C. ForPhu Lae extracts, the highest activity occurred at 60 ◦C. Thepeel seems to have the most promise for bromelain extrac-tion because it accounts for the largest waste proportion. Thepineapple waste residues after bromelain extraction containhigh amount of fiber. It would be useful to suggest furtherapplication of these residues for paper and paper board pro-ductions.

Acknowledgements

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

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