Elsevier

Biochemical Engineering Journal

Volume 52, Issues 2–3, 15 November 2010, Pages 205-211
Biochemical Engineering Journal

Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar

https://doi.org/10.1016/j.bej.2010.08.012 Get rights and content

Abstract

Extraction of bromelain from pineapple peel (Nang Lae cultv.) using aqueous two phase system (ATPS) was optimized. Some biochemical properties including collagen hydrolysis were also investigated. Bromelain predominantly partitioned to the polyethylene glycol (PEG)-rich phase. The highest enzyme activity recovery (113.54%) and purification fold (2.23) were presented in the top phase of 15% PEG2000–14% MgSO4. Protein pattern and activity staining showed the molecular weight (MW) of bromelain to be about 29 kDa. The extracted bromelain showed the highest relative activity at pH 7.0 and 55 °C. Its activity was decreased continuously by increasing NaCl concentration (up to 1.5% (w/v)). The bromelain extract was applied to hydrolyze the skin collagen of beef and giant catfish (0–0.3 units). The β, α1, α2 of giant catfish skin collagen extensively degraded into small peptides when treated with 0.02 units of the bromelain extract. Bovine collagen was hydrolyzed using higher bromelain up to 0.18 units. This study showed the ATPS can be employed to partially purify bromelain from Nang Lae pineapple peel and the enzyme effectively hydrolyzed the collagens.

Introduction

Plant proteases have received special attention in the field of medicine and biotechnology due to their properties. The best known plant proteases with commercial value are papain from Carica papaya [1], ficin from Ficus spp. [2] and bromelain from Ananas comosus [3]. Bromelain is a proteolytic enzyme normally present in pineapple fruit and stem [4]. It has been widely used in food, medical–pharmaceutical, cosmetic and other industries. In food industry, it has been used for meat tenderization [5], [6], [7], [8], beer clarification, protein hydrolysate production [9], [10], [11] and prevention of apple juice browning [12]. It has been known for clinical and therapeutic applications, particularly for modulation of tumor growth and third degree burns [3]. In cosmetic industries, bromelain is used as active ingredient to provide gentle peeling effect [10]. In addition, bromelain has also been used for skin pre-tanning, softening and bating in leather industries [13], for improving the dyeing properties of protein fibers, decomposing or partially solubilizing protein fiber from silk and wool [14], [15]. Furthermore, novel use of bromelain as hydrolyzing agent for the release of antimicrobial peptides of leatherjacket (Meuchenia sp.) insoluble proteins has been reported [16].

Collagen is the major determinate of the texture of mature and older slaughter animal meat. There is a reasonable correlation between total collagen content and the eating quality of meats [17]. Tenderization of those meats may be achieved using protease for degradation of connective tissue and myofibrillar proteins [13], [17]. Apart from food aspect, collagen has extensively been used in cosmetic applications. Hydrolyzed collagen is incorporated in skin regenerating product designed to counteract skin dryness as well as prevent skin damage [18], [19]. Moreover, it has been reported that collagen hydrolysate is beneficial for joint and bone health [20].

Thailand is the world's largest producer of processed pineapple such as canned pineapple and pineapple juice. Nang Lae cultivar is a pineapple cultivated only in Chiang Rai of Thailand [21]. It has exceptional juiciness and a vibrant tropical flavor that balances the tastes of sweet and tart, as a consequence consumption of this cultivar is very popular. Due to increasing demand and abundant availability year-round, wastes are proportionally increased, especially peel accounting for 30–42% of a whole fruit of pineapple [22]. Besides low economical utilization like animal feed, value addition of the pineapple wastes has been attempted using them for the production of bromelain and citric acid [4], [23]. Therefore, it is worth to extract the bromelain from Nang Lae pineapple peel and use this protease in the study of collagen hydrolysis for food and cosmetic applications.

Aqueous two phase system (ATPS) is an effective and economically attractive method as it is attractive for the separation and purification of mixture of proteins/enzymes [24], [25]. It can remove undesirable byproducts present in the system such as unidentified polysaccharides, pigments and interfering proteins that lower the activity of enzyme. Compared to other separation and purification methods, extraction using ATPS has many advantages including low cost, ease of scale-up, scope for continuous operation, and environment friendly thus, making it an attractive alternative for isolation of biomolecules [25]. ATPS has been used for the partitioning and recovery of various proteases such as papain [24], trypsin [26], chymosin and pepsin [27], [28], protease from fermentation broth [29], [30]. Bromelain from pineapple fruit was also extracted by ATPS comprising PEG–potassium phosphate [31] and two phase system containing PEO–PPO–PEO block polymer [32]. However, there are no practically up-to-date reports on the two phase partitioning of bromelain from pineapple peel as well as biochemical properties of the extracted enzyme including the ability of collagen hydrolysis.

Hence, the objectives of the present study were to partially purify the bromelain from pineapple peel (Nang Lae cultv.) by ATPS and to determine the biochemical properties of extracted bromelain including hydrolysis pattern of collagens. The result from this study would be useful for several applications, especially food and cosmetic industries.

Section snippets

Chemicals and raw materials

Polyethylene glycols (PEG2000, 4000, and 6000 Da), casein and bovine serum albumin (BSA) were purchased from Fluka (Buchs, Switzerland). Ammonium sulphate ((NH4)2SO4), magnesium sulphate (MgSO4), potassium hydrogen phosphate (K2HPO4) and trichloroacetic acid (TCA) were procured from Merck (Darmstadt, Germany). Collagen from bovine achilles tendon (C9879), beta-mercaptoethanol (βME), Coomassie brilliant blue G-250 were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

The pineapple peel (

Effect of PEG on bromelain partitioning

The bromelain partitioning in the ATPS containing different molecular weights (MW: 2000–6000 Da) and concentrations of PEG (12–18%, w/w) with 17% MgSO4 was studied. As shown in Table 1, partitioned bromelain strongly depended on the MW and the concentration of PEG. The result showed that the VR of the system ranged from 0.84 to 1.19. Distribution of the protein and protease in ATPS is reported by partition coefficient of protein (KP) and partition coefficient of enzyme (KE), respectively. High

Conclusions

The bromelain from the Nang Lae pineapple peel was extracted by the aqueous two phase system. The top phase from 15% PEG2000–14% MgSO4 provided the highest bromelain recovery. The extracted bromelain showed the optimum pH at a neutral. The highest activity of bromelain was found at 50–60 °C. Forty percentages of bromelain activity lost in the presence of 1.50% (w/w) NaCl. Based on collagen degradation, it is suggested that bromelain from Nang Lae pineapple peel could be used for meat

Acknowledgements

The authors would like to thank Mae Fah Luang University and National Research Council of Thailand (NRCT) for financial support under the project No. PK./2553-40.

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