Green technology based extraction and evaluation of bromelain and other bioactive peptides from pineapple industrial waste for their value addition | |
| Author | Thatchajaree Mala |
| Call Number | AIT Diss no.FB-21-01 |
| Subject(s) | Pineapple Bromelains Peptides Green technology |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of doctor of Philosophy in Food Engineering and Bioprocess Technology |
| Publisher | Asian Institute of Technology |
| Abstract | Pineapple (Ananas comosus) is considered one of the most widely produced fruits in tropical and sub-tropical countries. Thailand is regarded as one of the largest producers and exporters of pineapple globally. Canned pineapple and refined juice are the major processed products, and 80% of them are exported, whereas the remaining 20% are sold locally. Bromelain is one of the essential proteolytic enzyme complexes that are obviously available in pineapple. During pineapple processing, the crown, stem and core are separated, representing approximately 70% (w/w) of total pineapple weight. As a result, with increasing pineapple production, wastes from pineapples are also consequently increasing. Waste disposal is becoming a significant concern since it usually causes microbial spoilage and serious environmental issues. Bromelain and other cysteine proteases, well-known enzymes that are present in a variety of pineapple parts, including in crown, stem, peel, and core. On the other hand, bromelain proteases are normally unstable and sensitive under stress conditions in the presence of high temperature, organic solvents and chemicals, resulting in a reduction of enzymatic activity of this health-promoting enzyme, reducing their pharmacological, industrial and biotechnological applications. The conventional pasteurization method for pineapple juice has long been employed for microbial inactivation to ensure safety and for shelf life extension. However, conventional pasteurization has a detrimental effect on the sensory attributes and nutritional qualities of processed foods. The first objective of this study was to evaluate the effect of processing on bromelain in pineapple juice. Based on the bioactive characteristics of peptides and enzymatic activity, fresh juice showed the highest protein content (28.13 mg/ 100 mL) and enzymatic activity (62.66 U/ 100 mL) when compared with the commercial pasteurized pineapple juice (14.43 – 15.76 mg/ 100 mL and 33.01 – 35.29 U/ 100 mL). Furthermore, as the time spent at pasteurized temperature (93 °C) was increased from 1 – 5 min, the protein content and enzymatic activity significantly decreased from 9.72 to 2.39 mg/ 100 mL and 8.72 to 3.17 U/ 100 mL respectively. It is thus, evident that thermal processing or commercial pasteurization reduces the nutritional values, especially in protein content and enzymatic activity of pineapple juice. Therefore, there is a dire need to implement alternate food processing technology that can inactivate microorganisms in pineapple juice and retain the nutritional and sensory attributes. The second objective focused on alternative technology to preserve pineapple juice and retain nutritional and quality attributes. In this objective, fresh pineapple juice was subjected to thermosonication (TS) treatments with processing variables of temperature (25 - 65 °C), time (2 - 10 min) and amplitude (30 – 70%) at a constant frequency of 24 kHz. Response variables: total phenolic content (TPC), bromelain activity and microbial inactivation were optimized using response surface methodology (RSM). The optimized TS treatment conditions for juice were 62.33 °C, 2 min and 35.32% amplitude, which corresponded to bromelain activity of 63.24 U/ 100 mL of juice, TPC of 177.3 µg GAE/ 100 mL, total plate count 2.74 log CFU/ mL and yeast and mold count 2.301 log CFU/ mL. TS treated, pasteurized (95 °C for 1 min) and control (untreated) pineapple juices were stored at 4 ± 1 °C for 28 days and evaluated for the microbial count, nutritional quality and physicochemical attributes. Total phenolic contents, ascorbic acid and protein contents of TS treated juice were significantly higher than pasteurized juice after 28 days of storage. However, there was no significant difference in DPPH inhibition and total plate count of TS treated and pasteurized pineapple juice after the storage period. Thus, TS treatment of juices can serve as an alternative to conventional thermal pasteurization to obtain better nutritional and microbial attributes during the storage period. The third objective aimed to evaluate the role of extraction techniques, especially microwave- and ultrasonic-assisted extractions, on the enzymatic activity of the bioactive compounds from pineapple waste. Subsequently, the chemical and bioactive characteristics of the extracts from optimal conditions were also evaluated. In this objective, ultrasonic-assisted extraction (UAE) and microwave-assisted extraction (MAE) were used to extract bromelain and bioactive peptides from pineapple by products. Response surface methodology (RSM) was used for the optimization of extraction. Independent extraction parameters for UAE were ultrasonic amplitude (60 - 100%), extraction time (10 - 30 min) and solvent to material ratio (20 - 40 mL/ g) whereas, for MAE independent variables were microwave power (100 - 300 W), irradiation time (5 - 15 min) and feed to solvent ratio (1:8 - 1:12 g/ mL). The optimized extraction conditions were 99.96%, 26.83 min and 20.96 mL/ g for UAE and 100 watt, 8.99 min and 1:8 g/ mL for MAE. Under these optimal conditions, total sugar, protein content and proteolytic activity for UAE were 15.71 ± 0.03 mg/ mL, 4.01 ± 0.04 mg/ mL and 196.46 ± 3.29 U/ mL, respectively, whereas, the response variables were 33.87 ± 0.03 mg/ mL, 2.50 ± 0.01 mg/ mL and 154.08 ± 1.49 U/ mL for MAE, respectively. Scanning electron microscopy was used to observe changes in the morphology of the sample before and after extraction. Molecular weight determination showed that the major protein band in the extracts (UAE and MAE) was at ∼23 kDa, corresponding to bromelain. Furthermore, pineapple crown protein extract obtained by UAE showed higher proteolytic activity (> 80% relative proteolytic activity) in a pH range 4 - 9 and at the optimum temperature of 60 °C. The results indicated that bromelain and other bioactive peptides from pineapple by-products have potential in food, feed, and pharmaceutical products development. The last objective focused on investigation and formulation in optimized bromelain loaded pectin/ hi-maize starch hydrogel beads to enhance the stability, bioaccessibility and improve the effective delivery of bromelain. Herein, hi-maize starch was incorporated into pectin by the extrusion method. Five formulations were represented with different ratios. In this study, the ratio of pectin and hi-maize starch concentration significantly influenced encapsulation efficiency. The swelling behavior of hydrogels was examined and reported in three media (distilled water, simulated gastric (SGF, pH 1.2) and intestinal (SIF, pH 7.4)). The hi-maize starch improved swelling properties and also regulated the bromelain release behavior of hydrogels. In phosphate buffer (pH 7.4), the swelling of hydrogel beads was higher than in pH 1.2 and water. Furthermore, the SEM images showed that incorporating 1.5% (w/w) hi-maize starch resulted in more compact hydrogel beads than applying pectin alone. The presence of bromelain in the particles was confirmed by using Fourier transform-infrared (FT-IR) spectroscopy. In an in vitro study, encapsulated bromelain in pectin/ hi-maize starch hydrogels was more resistant to undesirable gastrointestinal conditions than pectin hydrogels alone. The thermal stability of the encapsulated bromelain in all treatments was significantly improved compared to the non-encapsulated bromelain. According to the results of this study, pectin and hi-maize starch are suitable as a carrier for controlled release of bromelain delivery applications. |
| Year | 2021 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Food Engineering and Bioprocess Technology (FB) |
| Chairperson(s) | Anal, Anil Kumar |
| Examination Committee(s) | Loc Thai Nguyen;Ekbordin Winijkul |
| Scholarship Donor(s) | Rajamangala University of Technology Krungthep (RMUTK), Thailand;Royal Thai Government |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2021 |