Bromelain extraction using single stage nanofiltration membrane process

Abstract

Pineapple (Ananas Comosus) is a tropical fruit having exceptional juiciness, vibrant tropical flavor and immense health benefits. Pineapples are not only taken fresh but they have been commercialized in the canning industry. Morris cultivar is mostly supplied to the canning industry where only the flesh is utilized and the rest of the pineapple (50 wt%) such as the core, stem, peels and crown are discarded as wastes. In the extraction of bromelain which is a vital proteolytic enzyme the whole pineapple including its peels, core, stem and crown can be utilized. This enzyme is very valuable and considered as a food supplement with a wide range of therapeutic benefits. Thus, in this study, bromelain is extracted from the flesh, crown, stem, core and peel of pineapples using simple one stage hollow fiber nanofiltration membrane process. The different parts of the pineapple were crushed to extract the juice. The juice was then centrifuged and the supernatant was then passed through a nanofiltration membrane. Results revealed the retentates from the different pineapple parts contain high amounts of bromelain in descending order flesh > peel > core > crown > stem. The amount of bromelain activity increased after every process especially after freeze drying.

Introduction

Pineapple (Ananas Comosus) is popular in the canning industry and juice products. Malaysia is one of the largest pineapple producers around the world and Johor is the state that produces most of the pineapple in Malaysia. In 2016, approximately 0.27 million tons of pineapples which accounted for 70% of total production were produced in Johor (MPIB 2021). Basically, pineapple industries create almost 50% (w/w) wastes (peel, core, stem and crown) of total pineapple weight. This means, increasing pineapple production also increases the pineapple wastes.

There are several cultivars in Malaysia like Morris which are used in canning industry and the waste are converted and sold as cheap fertilizers. Generally, about 50–60% of fruit contributes to waste like core, peel, stem and crown. Since the flesh is always eaten fresh and also mainly used in canning products, peel, core, crown and stem can be a very good source for bromelain enzyme rather than being thrown away. Fortunately, bromelain enzyme has been identified from pineapple waste as a potential source of valued materials. Bromelain is in high demand because of its applications in food, medical and cosmetic industries (Chaurasiya et al. 2015). Therefore, the current focus in this field is to design an effective concentration and purifying process employing wastes from the pineapple industry with the goal in reducing the number of procedures required to achieve the desired enzyme purity. Generally, bromelain does not necessitate high purity for most commercial applications except for research, pharmaceuticals and medical field, therefore the enzyme is typically generated in large quantities as crude for requests in high-volume manufacturing (Illanes 2008; Vasiljevic 2020).

Proteinases, peroxidases, phosphatases, protease inhibitors, and organically bound calcium are all found in bromelain. The ratio may vary depending on climate circumstances during plant growth, soil composition, geographical location where the pineapples are grown, and the type of pineapple variety (Tochi et al. 2008). Variations in bromelain's activity may also be due to management of the crop such as artificial flowering induction (hormone sprayed) or natural flowering. Artificial flowering induction (hormone sprayed) provides certain advantages but it could also result in poorer fruit quality compared with natural flowering (Husin et al. 2018). Since the pineapple was collected from the same place and same variety, the probable reason Morris cultivar with no crown shows higher bromelain activity after crushing/juicing due to the artificial flowering induction. In a recent study, it was found that the crown plays an important role in maintaining quality of the harvested pineapple (Liu et al. 2017).

Up to date, various techniques were applied in order to gain highest bromelain production such as reverse micellar extraction, precipitation, aqueous two-phase system and ultrafiltration. All these procedures require several processes. Hence, in this study polymeric hollow fiber nanofiltration membrane method with unique characteristics is proposed as a feasible separation technique to extract bromelain as a single step. Besides that, nanofiltration membrane method can be scaled up easily and offer continuous operation. Furthermore, this method attracts an immense attention in particular for the extraction of an enzyme in recent times.

Materials and methods

Materials

Chemicals

Chemicals used in this experiment included casein, _L-tyrosine, anhydrous disodium phosphate (Na₂HPO₄), L-cysteine, ethylene diaminetetraacetic acid (EDTA), Bradford reagent, trichloroacetic acid (TCA), sodium acetate and acetic acid. All chemicals used were of analytical grade and used as received without any further purification and were obtained from Sigma-Aldrich.

Raw materials

The Malaysian pineapples (Ananas comosus) species, Morris pineapple with no crown and partially ripened, Morris pineapple with no crown and fully ripened, Morris pineapple with crown and partially ripened were collected from a plantation in the Kg Parit Gantung, Benut, Johor, Malaysia as shown in Fig. 1.

Fig. 1.

a Morris pineapple with no crown and partially ripened (dark green colour), b Morris pineapple with no crown and fully ripened (50% in yellow colour), c Morris pineapple with crown and partially ripened (dark green colour)

Methods

Preparation of crude pineapple extract

All the pineapples collected were washed. The flesh, peels, core, stem and crown from the different species were segregated and labelled. They were chopped and crushed separately by using an extruder to obtain the juice. The resulting juice obtained was centrifuged (Shimadzu Hermle Labor Technik GmbH—Z323K) for 10 min at 3500 rpm to remove all suspended impurities. The obtained supernatant was collected for further experiments.

Molecular weight cut-off of hollow fiber nanofiltration membrane

Before passing through the hollow fiber (HF) nanofiltration (NF) membrane, the membranes were characterized in term of its pore size (MWCO). The molecular weight cut-off (MWCO) of the membrane must be determined so as to assess its suitability. The method used was as described by Idris et al. (2007) and in order to determine the concentration of PEG, a calibration curve was calculated. Also, a graph of the PEG rejection rates versus MWCO was plotted so as to determine the MWCO at 90% rejection (Idris et al. 2007).

NF of supernatant from centrifuge

The obtained supernatants were then filtered using the self-fabricated HF NF membrane. The self-made HF NF used were made from polyether sulfone (PES). The filtration process was conducted at a rate varied from 300, 450, 600 and 750 ml/min at varied pressures from 1, 2, 3 and 4 bar. The membrane module was operated under cross flow filtration setup in which the feed flows tangentially across the membrane surface. From the nanofiltration process, the permeate and retentate were obtained. The schematic diagram of the nanofiltration membrane system is depicted in Fig. 2. The system set up consisted of a feed tank and membrane module. The supernatant from the centrifuge (pineapple crude enzyme extract) which was the feed, was purified using NF. The purified bromelain which was the retentate was collected and the permeate which mostly consists of water and other small molecule sugars was accumulated separately. The bromelain extract from the retentate was stored in a refrigerator at 2 °C for further use. The characteristics and specifications of the nanofiltration membrane module used are reported in Table 1.

Fig. 2.

Schematic diagram of NF system for bromelain purification and nanofiltration membrane module

Table 1.

Membrane specifications

Type	Polyether sulfone (PES)
Configuration	Hollow fiber
MWCO (kDa)	2.8
Membrane module size (L X D)	Approximately 1.21 × 0.21 m
Total number of fiber	500
Membrane Specs (2 to 5 nm)	Can achieve rejection rates as high as 95 to 99% thus good at retain the bromelain at the retentate

After each experiment, the NF membranes were washed with water and reused again. The membrane can be repeatedly used for at least 6 cycles until the flux rate decreased to very low levels and the membranes were cleaned again according to the cleaning procedure outlined (Nor et al. 2016) with some modifications. In this procedure, the membranes were cleaned with 2% w/w sodium hydroxide (NaOH), 1% w/w nitric acid (HNO₃) and 2% w/w sodium peroxide (Na₂O₂). To ensure that the cleaning process has been completed, the water flux was measured. Filtrate flux rate of the membrane can be measured as:

$$A=\frac{V}{J\times T}$$ 1

where $A$ = membrane area (m²), $V$ = volume of filtrate generated (liters), $J$ = filtrate flux rate (liters/m2/hour (LMH)), $T$ = process time (hours).

Freeze drying of retentate bromelain solution

Various concentrations of maltodextrin (MD) in the range of 2–10% concentration (w/w %) were prepared to act as a carrier or a lyoprotectant and were dissolved in bromelain solution. The liquid sample containing bromelain with the dissolved MD was then freeze-dried using (FreeZone 2.5, Labconco) freeze dryer for 48 h to convert it into powder form. The drying temperature used was −51 °C. The freeze-dried sample was then crushed to obtain bromelain powder and placed in sealed plastic flask.

Analysis

pH and total soluble solids (TSS)

The pH of the samples was tested using a pH meter (Mettler Toledo) while the total soluble solids (TSS) was confirmed using a hand refractometer (Atago Pal-1) with units in ^°Brix. On the prism of the refractor, a drop of solution was squinted and the value of TSS was determined by direct reading of the instrument.

Total sugar

Total sugar in the samples was examined using the DNS method by mixing 1 ml of the diluted extract with 1 ml of 5% phenol. Then, 5 ml of a concentrated sulphuric acid (98%) was added and the mixture was allowed to stand for 10 min. The mixture was then placed in a water bath for 20 min at 20 °C. The colour developed was read at 480 nm using a spectrophotometer (Simadzu UVmini—1240) and concentration of glucose was determined from the standard curve of _D-glucose with a concentration range from 20 to 100 mg/l. (Nor et al. 2016).

Protein content

0.9 ml of distilled water was put into small sampling bottle. Then, 0.1 ml of each of samples were added into the sampling bottle. Then, 1.5 ml of Bradford reagent was added to all the sampling bottles. All the sampling bottles were then closed and kept in light off condition for 10 min. Then, protein content was analyzed using spectrophotometer at 595 nm and Bradford reagent as blank (Ketnawa et al. 2012).

Bromelain activity and bromelain specific activity

Casein digestion unit (CDU) was used to examine the enzyme activity of all samples. L-tyrosine and casein were used as a standard and substrate, respectively. The assay was based on proteolytic hydrolysis of the casein by the enzyme. Bromelain hydrolysed the casein to release L-tyrosine. One unit of enzyme activity is defined as the amount of enzyme, releasing a product equivalent to 1 µg of tyrosine/min/ml under the standard assay conditions and expressed as CDU/ml. The absorbance was read using spectrophotometer (Simadzu UVmini—1240) at wavelength 275 nm. The specific activity (SA) of the enzyme is calculated using Eq. (2) as CDU per mg of protein. (Nor et al. 2016)

$$SA\left(\frac{\mathit{CDU}}{\mathit{mg}}\right)=\frac{\mathit{EA}}{\mathit{PC}}$$ 2

where EA is the enzyme activity and PC is the protein content.

Results and discussion

Pineapple distribution weight

Figure 3a, b show the weight percentages before crushing of various pineapple parts obtained from 20 pineapples (partially ripened) for two Morris types (with crowns and without crowns), respectively. Weight percentage of flesh part of Morris pineapple without crown was higher than Morris pineapple with crown. The flesh of Morris pineapple with crown accounts for 45 wt% whereas the flesh of Morris pineapples without crown accounts for 52 wt%. The weight percentages of both Morris pineapple with and without crown parts were as follows; flesh > peel > core > crown > stem.

Fig. 3.

Weight before crushing and volume after crushing of pineapple with (a) crown (b) no crown

Total weight contribution of pineapple parts except flesh of Morris pineapple with crown is 55 wt% which is higher than the flesh weight percentage contribution (45 wt%). The juice obtained from flesh after crushing is 38 vol.% whilst the peels contributed to 34 vol.%, followed by core, crown and stem in reducing volume percentage. However, for the Morris pineapple without crown the flesh contributed to 52 wt% of the total weight and the volume of the juice obtained was also much higher (48 vol.%) compared to only 38 vol.% of juice obtained from flesh of Morris pineapples with crowns.

Figure 4a, b show the weights of the pineapple without crown at different ripeness. The difference in the degree of ripeness is illustrated in Fig. 1. Partially ripened fruit had higher mass in flesh compared to the fully ripened pineapple as shown in Fig. 4a, b. The juice extracted from a partially ripened fruit was also much higher (see Fig. 4a). Figure 4 also shows that the weight of core of fully ripened pineapple was higher than partially ripened pineapple. The result can be supported by findings in Nadzirah et al. (2013), where the percentage of pulp in pineapple core extracts was at highest as the maturity index increased. According to Brownleader et al. (1999), a fruit tends to has rigid and well-defined cellular structures prior ripening and as the fruit ripens, the cell walls become soft and diffused due to organic acids released.

Fig. 4.

Weight before crushing and volume after crushing of pineapple with (a) no crown (partially ripened—dark green colour) (b) no crown (fully ripened—50% in yellow colour)

High weight percentage of flesh in Morris pineapple without crown can be regarded to the effect of hormone spray by the local farmer. During cultivation of the Morris pineapple with no crown, the hormones were sprayed at the age of 7 to 8 months old to promote pollination and in the same time to produce evenly fruit size. Therefore, the Morris pineapple with no crown has evenly fruit size compared to pineapple with no crown. This is also the reason core part of pineapple without crown gained more weight than the pineapple with crown. In India, Morris pineapples were sprayed with ethephon hormone and planofix for duration of 10 months. As a result, the fruit produced had large and uniform size and maximum flowering (Verma et al. 2019). Besides, Husin et al. (2018) revealed that the production of pineapple fruits is unpredictable where natural flowering is used because it can lead to unsynchronized and uncontrolled harvesting. Thus, in order to ensure more synchronized and controlled fruiting throughout the years, flower inducers such as ethephon, calcium carbide, and acetylene have been used to induce pineapple flowering.

HF nanofiltration membrane characteristics

Molecular weight cut-off (MWCO)

Figure 5 illustrates the MWCO of the HF NF membrane used in this study. Figure 5 shows that the MWCO at 90% rejection is about 2.8 kDa. Nor et al. (2016) in their study have mentioned that membrane with smaller than 10 kDa molecular weight is suggested for improved enzyme retention. Therefore, this HF NF membrane can separate the bromelain enzyme with high retention rate. Thus, all molecules smaller than 2.8 kDa can pass through the HF NF membrane but the bromelain will be retained as retentate because bromelain enzyme's molecular weight (MW) is between 23.4 to 35.73 kDa (Arshad et al. 2014).

Fig. 5.

The MWCO of the nanofiltration membrane showing 2800 dalton at 90% rejection rate

Pressure and flow rate of the nanofiltration process

The supernatant from the centrifuged process was then passed through HF NF. Figure 6a, b illustrate the bromelain activity at retentate of the HF NF process at different flow rates and pressures. The results found that increasing the pressure reduced the bromelain activity while increasing the flow rate increased the bromelain activity with (p < 0.05). By increasing the transmembrane pressure, enzymatic activity decreased, due either to the rupture or modification of enzyme structures inside of the membrane pores or to a rude contact with fouling (Lopes et al. 2009). It can be observed that highest bromelain activity was obtained at a pressure of 1 bar and flow rate of 750 ml/min as depicted in Fig. 5. As acknowledged by Lopes et al. (2009), bromelain activity was decreased when membrane processing was performed at high trans-membrane pressure (TMP). Doko et al. (1991), Hebbar et al. (2012) and Nor et al. (2016) in their work also used low pressure in UF membrane processing range 1–3 bar. Whereas, Nor et al. (2017) discovered raising the flow rate can improve flux value as well as enzyme’s selectivity. Nor et al. (2016) also used high flow rate of 690 ml/min in their research to achieve high bromelain activity.

Fig. 6.

Bromelain activity of retentate at different (a) flow rate at constant pressure (1 bar) versus (b) pressure at constant flow rate (450 ml/min) of the HF NF process

Flux rate of the membrane

Figure 7 shows that the flux rate decreases with time and reaches the low point after 50 min. However, after cleaning with water, the membrane can be used again and retain its flux rate for another 50 min. Results shows that this HF NF have good permeation rate since it can be used for at least 6 times with intermittent backwashing before the flux rate decreased to very low values. After the 6th cycle, the membrane must be washed to attain good permeation rate again. The HF NF membranes were cleaned using 2% w/v sodium hydroxide (NaOH), 2% of methanol and 3% of sodium peroxide. The water flux was determined after cleaning to ensure that the foulants were removed. The results represented in Fig. 7 shows that the membrane maintains its high-water flux even after six times of fouling by pineapple fruit suspension (Khairuddin et al. 2019).

Fig. 7.

Water and pineapple juice for five sequential fluxes of the nanofiltration membrane at 1 bar

Generally, the disadvantage of membrane filtration is reduction of permeation rate over time due to the fouling deposition. The fouling elements in fruit juice that are mostly collected are polysaccharides and macromolecules, such as hemicelluloses, lignin, cellulose and pectins. The drawback caused by these foulants are decreasing in flux and variations of physicochemical properties of filtrate. Hence researchers have efficiently justified membrane fouling by restoring membrane flux recovery. Yet, the fouling is impossible to eliminate completely (Madaeni et al. 2001; Bhattacharjee et al. 2017).

Nor et al. (2016) detailed in their study that the permeate flux decreased significantly for the first 30 min and decline slowly until the end of the process. The continuing decrease in the flux rate was due to the consequence of the concentration polarization occurring due to proteins (including bromelain enzyme). In their high cross flow setup, the existence of the smaller solutes would attribute to a thin cake layer. Yet, the phenomenon could be minimised due to the smaller pore size in UF stage 2.

Physicochemical properties and specific activity of bromelain from Morris pineapple (core and flesh)

Table 2 tabulates the properties of the flesh pineapple extract from Morris with crown and no crown while Table 3 tabulates the properties of the core pineapple extract from Morris with crown and no crown. The original flesh and core of the Morris pineapple is characterized by an acidic pH value of 3.91 (flesh no crown), 3.90 (flesh with crown), 3.89 (core no crown) and 3.86 (core with crown). The pH is slightly changed (p < 0.05) in the retentate and permeate for both parts (flesh and core) demonstrating the pH stability of the extract during the process. Beside pH, it is also characterized by total soluble solid (TSS) of 7.7°Brix (flesh no crown), 7.6°Brix (flesh with crown), 4.1°Brix (core no crown) and 3.9°Brix (core with crown) at the feed. The TSS values were slightly changed in the permeates but significantly higher at retentates in both parts (flesh and core) compared to the TSS values in feed. Meanwhile, total sugar was also characterized with value of 43.21°Brix (flesh no crown), 40.45°Brix (flesh with crown), 38.61°Brix (core no crown) and 30.15°Brix (core with crown). In contrast, total sugars decreased in the permeate in both parts (flesh and core) with 16–38% reduction but slightly change in the retentate, demonstrating the polysaccharide was released in the permeate and some is still retained in the retentate.

Table 2.

Properties of the flesh pineapple extract from Morris cultivar with crown and no crown (Pressure at 2 bar; Flow rate at 750 ml/min)

Table 3.

Properties of the core pineapple extract from Morris cultivar with crown and no crown (Pressure at 2 bar; Flow rate at 750 ml/min)

The protein content of the flesh at the feed was 0.98 mg/ml (no crown) and 0.96 mg/ml (with crown) while the protein content of the core at the feed was 0.55 mg/ml (no crown) and 0.58 mg/ml (with crown). While the bromelain activity at the feed of the flesh was 251.87 CDU/ml (no crown) and 219.47 CDU/ml (with crown) which is higher than core at the feed which is 218.00 CDU/ml (no crown) and 186.22 CDU/ml (with crown). Thus, resulting high specific activity of the flesh was 257.01 CDU/mg (no crown), 228.62 CDU/mg (with crown) and core 396.36 CDU/mg (no crown) and 321.07 CDU/mg (with crown). In brief, the trait retention in the juice is reflected clearly in Table 2 and 3. In terms of pH the juice obtained from the flesh and core is the same. As expected, the TSS, glucose and protein are higher in the flesh as this part is mostly consumed than the core. In fact, the bromelain activity in the flesh is also higher. Studies (Pavan et al. 2012) have shown that fruit bromelain and stem bromelain contain different enzymatic composition.

Some reports have mentioned different bromelain enzyme activity even though they used the same pineapple’s variety like Smooth Cayenne and parts. Nor et al., (2016) have studied the bromelain enzyme activity of pineapple variety Smooth Cayenne and reported that the bromelain activity was 129.4 CDU/ml with the protein content of 1.71 mg/ml, resulting in a low specific activity of 75.3 CDU/mg protein. For pineapple core extract, researchers have found different bromelain enzyme activity such as reported by Chaurasiya and Hebbar, (2013) (129.41 CDU/ml), Hebbar et al., (2012) (415.12 CDU/ml) and Hebbar et al., (2008) (319.7 CDU/ml) even though all reports followed the similar sample preparation procedure. Although the same pineapple’s variety were used, the differences in the bromelain enzyme activity maybe because of dissimilarity in agricultural management used at various plantation. Additionally, the climate, soil and light levels also affected the metabolism of the pineapple fruit (Silvestre et al. 2012). Moreover, there are other factors that might also influence the bromelain enzyme activity such as age, fruit’s maturity and harvesting time of the pineapple (Bresolin et al. 2013; Chaurasiya and Hebbar 2013; Heinicke and Gortner 2019).

Table 4 depicts the recovery for enzyme content after undergoing NF membrane processing. NF membrane managed to retain more than 90% of enzyme activity for flesh with crown while for core with crown the enzyme activity was retained at 85.9%. While for pineapple Morris with no crown, flesh can retain higher enzyme activity than core with 74.6% compared to 57.6%. This phenomenon of enzyme loss for all the species before and after nanofiltration could be caused by the oxidation of enzymes and the resulting conformational changes to proteins. Furthermore, NF retentate (> 50%) showed significantly higher enzyme activity in accordance with the mass balance in Table 3, indicating the filtration process was successful in retaining the enzyme. It might be possible to retain more enzyme if the cross-flow velocity is reduced (Nor et al. 2016).

Table 4.

Mass balance of recovery of bromelain enzyme content during NF membrane processing

	Feed	Permeate		Retentate		Total (%)
Core no crown
Volume (ml)	950	550	57.9%	400	42.1%	100.0
Enzyme (CDU)	207,100.0	15,790.5	7.6%	103,480	50.0%	57.6
Core with crown
Volume (ml)	950	540	56.8%	410	43.2%	100.0
Enzyme (CDU)	176,909.0	24,575.4	13.9%	127,317.3	72.0%	85.9
Flesh no crown
Volume (ml)	950	510	53.7%	440	46.3%	100.0
Enzyme (CDU)	239,276.5	23,393.7	9.8%	154,968.0	64.8%	74.6
Flesh with crown
Volume (ml)	950	530	55.8%	420	44.2%	100.0
Enzyme (CDU)	208,496.5	34,863.4	16.7%	150,053.4	72%	98.7%

Bromelain activity

Figure 8a, b show the bromelain activity obtained from pineapples without crowns and with crowns, respectively. At early step of after crushing/juicing, pineapple without crown shows higher bromelain activity for flesh, peel and core. The bromelain activity increased after each process from crushing to NF process. The flesh, peels, crowns, core and stem from pineapples with crowns showed higher bromelain activity than pineapple without crown in retentate (Fig. 8a). From Fig. 8, it is observed that the flesh presented the highest bromelain activity followed by peel, core, crown and stem. This is in agreement with an earlier study by (Hale et al. 2005) that revealed pineapple fruit has a greater proteinase activity than stem bromelain.

Fig. 8.

Bromelain activity of each part of pineapple with (a) crown (b) no crown; after crushing, after centrifuge and after filtration (retentate) (Pressure at 2 bar; Flow rate at 750 ml/min)

Figure 9a, b show the bromelain activity obtained from partially ripened and fully ripened Morris pineapples without crowns. The results revealed partially ripened fruits have higher bromelain content compared to the fully ripened fruits. Bromelain activity was slightly higher in partially ripened flesh (275.5 CDU/ml) compared to fully ripened flesh (251.87 CDU/ml) after the sample was centrifuged. The results demonstrated that there is less bromelain content as well as bromelain activity in fully ripe fruits. This is because mature fruit changes morphologically, physiologically, and biochemically, all of which are fundamental to the quality of any cultivar, More ever, during ripening the process of senescence takes place (Hajar et al. 2012). Furthermore, fruits that are unripe have a tendency to contain higher levels of protease (Ramli et al. 2018). According to Amid et al. (2011), the commercially available form of bromelain in an aqueous extract was obtained from pineapple stems and unripe fruits. Even though the same pineapple’s variety of Morris was used, the bromelain activity of the same pineapple variety may have variances possibly caused by the difference in agronomic management. Factors such as age, fruit’s ripeness and harvesting time affect the bromelain activity of the pineapple. Bromelain activity was increased after each step. Figures 8 and 9 clearly represent the bromelain activity was highest after filtration (retentate) for all pineapple parts.

Fig. 9.

Bromelain activity of each part of pineapple with (a) no crown (partially ripened) (b) no crown (fully ripened); after crushing, after centrifuge and after filtration (retentate) (Pressure at 2 bar; Flow rate at 750 ml/min)

Nor et al. (2016) have reported in their research the bromelain activity using two-stage ceramic ultrafiltration process. The bromelain activities obtained in the first stage when using the 75 kDa ultrafiltration membrane were 129.4 CDU/ml, 125.2 CDU/ml and 133.3CDU/ml in the feed, permeate and retentate, respectively. Whereas, the bromelain activities in the second stage when using the 10 kDa ultrafiltration membrane were 124.5 CDU/ml, 45.5 CDU/ml and 485.4 CDU/ml in the feed, permeate and retentate, respectively. Chaurasiya and Hebbar (2013) in their study mentioned that the amount of bromelain in fruit varies with its maturity. A selection of partially ripe (suitable for processing) and fully ripe (perfectly soft) fruits were used to determine the bromelain content present in each fruit. Partially ripe fruits had a marginally higher bromelain enzyme activity (128.74 CDU/ml) than fully ripe fruits (119.69 CDU/ml). The results indicated that bromelain content and activity decreased as the fruit ripened which is in agreement to our study.

Freeze-drying of the bromelain juice (retentate)

Figure 10 shows the freeze-dried bromelain from flesh which was mixed with various percentage of MD. The percentage of MD was varied from 2 to 10% w/v and was added before the freeze-drying process. It was observed that the lower percentage showed that the powder is a bit sticky and difficult to handle as we can see in Fig. 9a–d. However, the highest percentage of MD added which is 10% (w/w) exhibits a powder with glassy appearance as illustrated in Fig. 9e. This result is in agreement with Sanchez et al. (2013) which added 20% (w/w) MD to red wine before freeze drying and their findings showed that most of alcohol and water were removed leaving behind the glassy powder which can be simply crushed.

Fig. 10.

Freeze dried bromelain powder with MD as encapsulation material. a 2% MD, b 4%MD, c 6% MD, d 8% MD, e 10% MD

Figure 11 shows that freeze dried bromelain activity obtained is higher (412.42 CDU/mg) compared to the bromelain activity of retentate (298.86 CDU/mg) after nanofiltration. The result is in agreement with (Devakate et al. 2009) who found that lower drying temperature from freeze drying can produce bromelain powder with higher enzyme activity. Moreover, freeze drying decreases the possibilities of protein denaturation.

Fig. 11.

Freeze drying of bromelain pineapple juice to powder

Conclusion

Flesh contributes the largest portion in terms of weight and contributes to the largest volume of pineapple juice. Meanwhile, peel showed the highest waste portion compared to the other parts and contributes to the second largest volume of juices. While Morris pineapple with crown showed higher bromelain activity than Morris pineapple with no crown (at retentate). Flesh of the pineapple exhibited the highest bromelain activity, while peel and core of the pineapple showed the highest bromelain activity among the other pineapple waste fractions. HF NF membrane process can be effectively utilised for crude bromelain extraction from pineapple fruit itself and also its waste. Bromelain activity from all parts of the pineapple were also increased at every step of crushing/juicing, centrifugation and NF process for both cultivars. Bromelain activity for Morris cultivar with crown is higher at retentate compared to Morris cultivar with no crown. Production of bromelain powder using HF NF and freeze-drying methods can retain the enzyme activity and decrease the risks of protein denaturation. The bromelain solution from flesh of Morris pineapple obtained was mixed with 10% MD and freeze-dried to obtain crude bromelain powder. After freeze drying the bromelain activity is retained with 412.42 CDU/mg. Further studies are needed to improve the purity of bromelain using HF NF, scale up of the process of extraction and also to extend the application to use as daily diet supplement or other food application.

Biological, chemical and microbiological

BaCl₂

Barium chloride

DNS

3,5-Dinitrosalicylic acid

EDTA

Ethylene diamine tetra acetic acid

HCl

Hydrochloric acid

HNO₃

Nitric acid

I₂

Iodine

KI

Potassium iodide

MD

Maltodextrin

Na₂HPO₄

Anhydrous disodium phosphate

Na₂O₂

Sodium peroxide

NaOH

Sodium hydroxide

PEG

Polyethylene glycol

PES

Polyether sulfone

TCA

Trichloroacetic acid

Instrumental techniques

ATPS

Aqueous two-phase system

HF NF

Hollow fiber nanofiltration

MWCO

Molecular weight cut-off

TSS

Total soluble solid

UV

Ultra violet

Vis

Visible

Unit

CDU

Casein digestion unit

Appendix

See Figs. 6, 7, 8, 9, 10, 11 and see Tables 2, 3, 4.

Authors' contributions

EM: Collection of data, chemical analysis, manuscript writing. AI: Conceive the idea, overall guidance, statistical analysis, conducting experiment, manuscript writing. HY: Statistical analysis, manuscript writing.

Funding

This work was supported by Malaysian Pineapple Industry Board (MPIB) and UTM for grant research R.J130000.7646.4C152 “Extraction of Bromelain from Pineapple Pulp and Fruit using Membrane Filtration”.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval

All authors approve to send this manuscript to JFST_.

Consent to participate

All authors have consented to the submission of the manuscript to JFST.