Retention of the Enzymatic Activity and Product Properties During Spray Drying of Pineapple Stem Extract in Presence of Maltodextrin

Abstract

The aim of this work was to investigate the effects of drying parameters on the retention of the enzymatic activity and on the physical properties of spray-dried pineapple stem extract. A Box and Behnken experimental design was used to investigate the effects of the processing parameters on the product properties. The parameters studied were the inlet temperature of drying gas (Tgi), the feed flow rate of the pineapple extract relative to evaporative capacity of the system (W_s /W_max), and the concentration of maltodextrin added to the extract (MD). Significant effects of the processing parameters on the retention of the proteolytic activity of the powdered extract were observed. High processing temperatures lead to a product with a smaller moisture content, particle size, and lower agglomerating tendency. A product with insignificant losses of the proteolytic activity (≈ 10%) and low moisture content (less than 6.5%) is obtained at selected conditions.

Keywords:

• Spray drying
• Pineapple extract
• Proteolytic activity
• Dried extract

INTRODUCTION

Pineapple (Ananas comosus) is a plant widely cultivated in almost all tropical and subtropical countries. It is native to South America where wild relatives still occur. Pineapple juice has great economic interest, being one of the most important non-citrus fruit juices sold in the world. It is important also as a source of proteolytic enzymes, mainly bromelain, a group of sulfur-containing proteolytic (protein-digesting) enzymes. Bromelain is used in the food industry for meat softening and in the beer production. It has also a recognized pharmacological action, demonstrated in vitro and in vivo, as antiedematous, anti-inflammatory, antithrombotic and fibrinolytic.[1,2]

The pineapple stem is one of the main wastes generated by the industrial processing of pineapple. This sub-product has high nutritional value, and may be used as a source for the extraction of bromelain and for preparation of fiber food products.[3]. In this work, it is proposed to use this material for the development of a bromelain loaded spray-dried extract, which can be used as nutraceutical, flavouring agent in ice cream, sweets, confectionery, or additive in soft drink concentrates, milk-based products, and baby foods.

A spray dryer is a piece of equipment wherein liquid feed is rapidly transformed into a dried particulate form by atomizing it into a hot drying environment.[4] The reduction of the degradation of thermally labile proteins and of the powder stickiness during spray drying of pineapple stem extract are typical challenges inherent to this process. The powder stickiness are mainly due to the presence of low molecular weight sugars, such as fructose, glucose, sucrose and some organic acids present in the fruit. In order to overcome these problems, drying aids such as isolated protein, maltodextrin with different dextrose equivalent (DE) are added to produce non sticky, free flowing powders.[5–8] Although the effects of adding these drying carriers on product stickiness are relatively understood, their effects on enzymes stability during spray drying is not well known. The purpose of this work was to evaluate the efficacy of the maltodextrin on the preservation of the crude enzymes stability and on physical properties of spray-dried pineapple stem extract.

MATERIALS AND METHODS

Materials

Pineapples (Ananas Comosus), variety Perola, produced in Frutal, Minas Gerais State, Brazil, were purchased from a local market (CEASA, Ribeirão Preto – SP, Brazil). Fruits were selected based on maturity, judged by colors (yellowish-greenish color). The stems were removed by hand, using a knife. The stems were pressed to squeeze out juice. The juice was filtered, added with 3 g/L of sodium benzoate, 0.5 g/L of cistein chloridrate and 2 g/L of EDTANa₂, and maintained under refrigeration (4°C) for the subsequent tests. The filtered juice was characterized by the determination of the solids, protein, reducing sugars content, and relative enzymatic activity.

Spray Drying

The drying was conducted in a bench-top spray dryer (model SD-05, Lab-Plant, Huddersfield, U.K), with concurrent flow regime. The drying chamber has 215 mm in diameter and height of 500 mm. The main components of the system were a feed system of the drying gas, constituted by a blower and an air filter, a temperature control system of the drying gas, and a product collect system (cyclone). The extract was fed to the spray dryer through a feed system, constituted by a peristaltic pump, a two fluid atomizer (inlet orifice diameter of 1.0 mm) and an air compressor.

The drying operation started with injection of the drying air into the SD-05 spray dryer. The air was heated to the desired temperature and then the pineapple extract was fed in at a preset flow rate together with the atomizing air. Maltodextrin (MorRex® 1914) kindly donated by Corn Products of Brazil was used as the drying aid, in order to evaluate its efficacy on the improvement of the stability of the crude enzymes during spray drying. Measurements of the outlet gas temperature, T_go, were taken at regular intervals in order to detect the moment when the dryer attained the steady state (± 15 minutes). Once the steady state was attained, samples of the powdered product were collected. The samples were used for the physical and chemical characterization of the product, through determination of its moisture content (≈ volatiles content), size distribution, particle morphology, protein content, and relative enzymatic activity.

Physical and Chemical Characterization of the Pineapple Stem Extract

The total reducing sugars in the crude pineapple stem extract was measured according to IAL (1985), [9] method 4.13.2. The total protein content was determined by the Bradford method,[10] using bovine albumin (BSA) as standard. The protein retention ratio in the spray-dried extract was defined as the ratio between the protein content in the product relative to crude extracts, as defined by Eq. (1):

(1)

where R_TP is the total protein retention ratio; T_P,SD is the total protein content in the spray-dried extracts; and T_P,EX is the total protein content in the unprocessed stem extract (dry basis).

The relative proteolytic activity of the extracts during spray drying was determined by the enzymatic hydrolysis of the casein and the quantification of the peptides formed by spectrophotometry at wavelength of 280 nm[11] (Murachi, 1976), using a spectrophotometer UV-VIS HP 8453 running the software HP Chem-Station®. A unity of relative enzymatic activity was considered as the amount of the stem extract (dry basis) able to produce an increment in the absorbance reading equivalent to the release of 1 μg of tyrosine per minute under the conditions of the assay (pH 7.2 and 35°C). The reference curve was constructed with tyrosine at final concentrations ranging from 10 to 100 μg/mL. The effect of the spray drying on losses of the relative enzymatic activity was estimated through definition of the retention of the enzymatic activity ratio, R_AE. This parameter was defined as the ratio between the relative enzymatic activity in the dried extract relative to crude extract (dry basis), according Eq. (2):

(2)

where A_P,SD is the relative proteolytic activity in the spray-dried extract; and A_P,EX is the relative proteolytic activity of the unprocessed stem pineapple extract (dry basis). The moisture content of the spray-dried extract was determined by the oven drying method[12]. Powder samples with a pre-defined mass, m_i, were placed in an oven heated at 102°C until constant mass, m_f. The percentage moisture content estimated by Eq. (3):

(3)

The size distribution of dried product was determined by optical microscopy and image analysis. A powder sample was dispersed on the surface of a microscopic lamina. Images of the powder were obtained with the aid of an optical microscope (Olympus® BX60MIV), connected to an image analysis system (Image Pro-plus® 4.5).[13] The particles morphology was determined by scanning electronic microscopy (S.E.M.) in a DSM 960 Zeiss, West Germany.

Experimental Design

Three factors and three levels Box and Behnken design[14,15] was used to investigate the effects of the processing parameters on the product properties. The parameters studied were the inlet temperature of drying gas, T_gi (70, 115, and 150°C), the feed flow rate of the pineapple extract relative to evaporative capacity of the system, W_s/W_max (24, 36, and 48%) and the concentration of the maltodextrin added to the extract, MD (60, 80 and 100% relative to the solid content). These conditions were chosen based on preliminary tests.[16] The feed flow rate of the drying air was maintained constant at 0.0227 m³/h.

The Box-Behnken design[14,15] is an independent quadratic design in that it does not contain an embedded factorial or fractional factorial design. In this design, the parameters combinations are at the midpoints of edges of the process space and at the center. These designs are rotatable (or near rotatable) and require three levels of each factor. For three factors, the Box-Behnken design offers advantages over the Box-Wilson Central Composite Designs in requiring a fewer number of runs.[17] This design allows the construction of a second-order polynomial model, which can be used to characterize or optimize a process. The resulting model has the following form:

(4)

where a₀ to a₉ are the regression coefficients; X₁ to X₃ denotes the factors; Y is the relative average or expected response associate with the combination factors; and E represents the experimental error. Before the drying runs, the evaporative capacity of the spray dryer was determined using water as standard liquid material. The evaporative capacity was defined as the feed flow rate that saturates the drying gas at system outlet gas temperature.[18–19] Table 1 present the results obtained. As expected, the evaporative capacities of the SD-05 spray dryer increases with inlet gas temperature. The results of evaporative capacity were used in the definition of the feed flow rate of the pineapple extracts fed to the dryer, according the Box-Behnken design.

Table 1 Water evaporative capacity of the spray drying obtained by the saturation criterion.[18]

Tgi (°C)	Tgo (°C)	Wmax (g/min)
70	39	9.30
110	58	12.3
150	76	16.0
Tgi: Inlet temperature; Tgo: outlet temperature; Wmax: evaporative capacity.

RESULTS AND DISCUSSION

The crude pineapple stem has a solids concentration of 11.46 ± 0.04%, total reducing sugars of 5.05%, total protein content of 203.5 ± 7.9 μg.mL⁻¹, and a relative enzymatic activity of 10965.2 ± 147.3 μg tyr.g⁻¹ . min⁻¹. This extract was submitted to spray drying, according to the experimental Box Behnken design. Samples of the dried product were withdrawn during the tests, and used for their physical and chemical characterization. Table 2 presents the processing conditions and the experimental results of the protein retention ratio, R_TP, retention of the enzymatic activity ratio, R_AE, product moisture content, U_P, and mean powder diameter, d_p.

Table 2 Processing conditions and experimental results of the protein retention ratio, R_TP, retention of the enzymatic activity ratio, R_AE, and product moisture content, U_P

Processing parameters				Experimental results
Tgi (°C)	WS/Wmax (%)	MD (%)	Tgo (°C)	RTP ( − )	RAE ( − )	Up (%)	dp (μm)
70	24	80	48	1.07	0.70	7.41	31.5
150	24	80	85	0.98	0.65	2.63	16.0
70	48	80	46	1.07	0.75	8.57	74.1
150	48	80	80	0.97	0.70	3.93	12.0
70	36	100	47	0.51	0.72	9.54	48.4
150	36	100	82	0.32	0.67	3.16	17.7
70	36	60	47	0.42	0.78	10.23	86.3
150	36	60	80	0.18	0.74	4.06	32.7
110	24	100	69	0.45	0.67	5.12	20.1
110	48	100	62	1.05	0.72	3.95	28.4
110	24	60	70	0.69	0.74	5.21	32.5
110	48	60	61	0.60	0.77	4.62	38.6
110	36	80	67	0.70	0.81	6.50	29.3
110	36	80	63	0.75	0.79	4.86	31.4
110	36	80	63	0.74	0.80	4.52	16.9

Analysis of variance (ANOVA) was carried out for the experimental results presented in Table 2, in order to identify the processing parameters presenting statistical significance on total protein retention ratio, R_TP; retention of relative enzymatic activity ratio, R_AE; and product moisture content, U_P. 3–5). show the ANOVA analysis results, respectively for the total protein retention ratio, R_TP; retention of relative enzymatic activity ratio, R_AE; and product moisture content, U_P. . ANOVA results revealed that the parameters W_S/W_max and MD were statistically significant at α level lower than 0.01, both for R_AE and R_TP. The inlet gas temperature, T_gi, was highly significant for R_AE and U_P at a α ≤ 0.01, and only at a α level lower than 0.1 for R_TP.

Table 3 ANOVA results for the total protein retention ratio, R_TP

Variable	Sum of squares	Degrees of freedom	Mean square	FCALC
Tgi	0.05013	2	0.02507	2.952
Tgi(L)	0.04805	1	0.04805	5.660∗∗∗
Tgi(Q)	0.00208	1	0.00208	0.245
Ws/Wmax	0.40053	2	0.20027	23.588∗
Ws/Wmax (L)	0.03125	1	0.03125	3.681
Ws/Wmax (Q)	0.36928	1	0.36928	43.496∗
MD	0.47328	2	0.23664	27.873∗
MD(L)	0.02420	1	0.02420	2.850
MD(Q)	0.44908	1	0.44908	52.895∗
Interaction effects
Tgi (L) Ws/Wmax (L)	2.510^−5	1	2.510^−5	0.003
Tgi (L) MD(L)	0.00063	1	0.00063	0.074
Ws/Wmax (L) MD(L)	0.11903	1	0.11903	14.019∗∗
Error	0.04245	5	0.00849	–
Total	1.15360	14	–	–
∗term is significant (α  = 1%); ∗∗term is significant (α = 5%); ∗∗∗term is significant (α = 10%); L = linear effect; Q = quadratic effect.

Table 4 ANOVA results for the retention of the enzymatic activity ratio, R_AE

Variable	Sum of squares	Degrees of freedom	Mean square	FCALC
Tgi	0.01329	2	0.00664	120.795∗
Tgi (L)	0.00451	1	0.00451	82.045∗
Tgi (Q)	0.00878	1	0.00878	159.545∗
Ws /Wmax	0.01375	2	0.00687	124.983∗
Ws/Wmax (L)	0.00405	1	0.00405	73.636∗
Ws/Wmax (Q)	0.00970	1	0.00970	176.329∗
MD	0.00990	2	0.00495	89.956∗
MD(L)	0.00781	1	0.00781	142.045∗
MD(Q)	0.00208	1	0.00208	37.867∗
Interaction effects
Tgi (L) Ws/Wmax (L)	0.00000	1	0.00000	0.000
Tgi (L) MD(L)	0.00003	1	0.00003	0.455
Ws/Wmax (L) MD(L)	0.00010	1	0.00010	1.818
Error	0.00028	5	0.00006	–
Total	0.03496	14	–	–
∗term is significant (α  = 1%); L = linear effect; Q = quadratic effect.

Table 5 ANOVA results for the product moisture content, U_p.

Variable	Sum of squares	Degrees of freedom	Mean square	FCALC
Tgi	65.49445	2	32.74723	28.335∗
Tgi (L)	60.33511	1	60.33511	52.205∗
Tgi (Q)	5.15934	1	5.15934	4.464∗∗∗
Ws/Wmax	2.66913	2	1.33456	1.155
Ws/Wmax (L)	0.06125	1	0.06125	0.053
Ws/Wmax (Q)	2.60788	1	2.60788	2.256
MD	0.96365	2	0.48183	0.417
MD(L)	0.69031	1	0.69031	0.597
MD(Q)	0.27334	1	0.27334	0.237
Interaction effects
Tgi (L) Ws/Wmax (L)	0.00490	1	0.00490	0.004
Tgi (L) MD(L)	0.01103	1	0.01103	0.010
Ws/Wmax (L) MD(L)	0.08410	1	0.08410	0.073
Error	5.77864	5	1.15573	–
Total	75.57549	14	–	–
∗term is significant (α = 1%); ∗∗∗term is significant (α = 10%); L = linear effect; Q  = quadratic effect.

Second order polynomial models relating the total protein retention ratio, R_TP, the retention of relative enzymatic activity ratio, R_AE, and the product moisture content, U_p, with the processing parameters were fitted to experimental results by non-linear regression, using the least squares method. Only the parameters presenting significant effects at a α level lower than 0.05 were used in the model. Models with high determination coefficient, able to adequately describe the effects of the processing parameters on the experimental responses, were obtained (see Table 6. Figures 1a–c present comparisons between the experimental results of the retention of the enzymatic activity ratio with the estimates obtained by Equation 5 (Table 6). These figures confirm the optimal agreement between the experimental data and the fitted model, exhibiting a root mean square error (RMSE) of only 0.005. As expected, these graphs show the existence of curvature effects of the processing parameters on R_AE, evidencing the existence of a maximum region. This region is delimited by the maltodextrin concentrations (60 to 80%), W_s/W_max ratio of 36% and temperature of 110ºC. The enzyme denaturation during spray drying of the pineapple stem extract has a complex functional relationship with the maltodextrin content and spray drying parameters. In general, the loss of the solvatation water during drying takes an important role in the protein degradation.[20] Therefore, the formation of hydrogen bonds between the –OH groups of the maltodextrin (MD) and the polar groups of the protein may contribute to maintain the protein conformation (stability) during drying.

Table 6 Models fitted to the experimental results of R_AE, R_TP, and U_p (dry basis)

Fitted models	R^2
(5)	0.989
(6)	0.920
(7)	0.872

Figure 1 Comparison between the experimental results of R_AE with estimates obtained by Equation 5 (a: MD = 60%; b: MD = 80%; c: MD = 100%).

The effects of the W_S/W_max ratio and concentration of the maltodextrin added to the extract, MD, on total protein retention ratio is presented in Fig. 2. The RMSE between the experimental and calculated values of R_TP was 0.08, confirming the adequacy of the Equation 6 (Table 6) to estimate the experimental results of total protein retention ratio obtained in this work. It can be observed that the maximum of total protein retention ratio is observed at maltodextrin concentrations of 80%, for the W_s/W_max ratio of 36% and T_gi of 110°C, defined previously.

Figure 2 Comparison between the experimental results of R_TP with estimates obtained by Equation 6.

Moisture content (U_P) is an important property of dried products, being an indicator of the drying efficiency. In general, the reduction in the product moisture content (and water activity as well) increases the product stability and shelf life. As can be observed in Figure 3, the product moisture content is inversely related to the inlet temperature of the drying gas. The dotted line in Fig. 3 correspond to the fitting of the experimental results of U_P with T_gi, given by Equation 7 (Table 6). For the spray-dried extract obtained at optimized conditions Equation 7 estimated a moisture content around 5.0%, which agrees with the experimental values.

Figure 3 Moisture content of the dried extract of pineapple stem as a function of the outlet gas temperature.

The product size was determined by optical microscopy and image analysis. In general, the powdered extract presented a wide size distribution, with mean diameters varying from 12 to 74 μm. Fig. 4a,b present results of the product size distribution obtained at four distinct operating conditions. The dashed lines in the Figs. 4a,b represent the data fitting by Rosin-Rammler-Bennet (RRB) model. Statistical analysis performed for the mean diameter values obtained for all experimental runs (see Table 2), indicate a significant effect of the drying gas temperature. Higher relative humidity conditions, which are often related to low drying temperatures, result in higher product moisture content during drying. Since the product has high sugar content, the increase in the moisture content, can cause the dissolution of small proportion of the sugar present, increasing the agglomeration tendency (stickiness) of the product.

Figure 4 Typical results of the product size distribution as function of the inlet gas temperature, T_gi: (a): Ws/W_max = 24%, MD = 80% (b): Ws/W_max = 48%, MD = 80%.

Figure 5 shows typical photomicrographs obtained by scanning electronic microscopy (S.E.M.), obtained at W_s/W_max ratio of 36%. These figures confirm that low drying gas temperatures lead to formation of agglomerated products. This behavior can be partly explained by the increase in the product moisture content, as discussed beforehand. Particles nearly spherical were obtained at temperatures of 110 and 150°C. The interaction between the maltodextrin and the extract resulted in a system of smooth (Fig. 5c), wrinkled (Fig. 5a) and dimpled (Fig. 5d) microparticles. It has been reported that sucrose can provides the formation of smooth particles when spray dried with maltodextrin and protein. The pineapple stem extract has about 50% of sucrose and probably an favorable ratio maltodextrin: sucrose and adequate drying temperature was reached in the processing conditions used in Fig. 5c.[21]

Figure 5 SEM: a) 150°C, 36% Flow rate and 100% MD, b) 70°C, 36% Flow rate and 60% MD, c) 150°C, 36% Flow rate and 60% MD, d) 110°C, 36% Flow rate and 80% MD.

CONCLUSION

The spray drying conditions, and the proportion of maltodextrin, have significant impact on preservation of the enzymatic activity, as well as on the physical properties of the spray-dried extracts of the pineapple stem. These results confirm the protective effect of maltodextrin against protein denaturation. Furthermore, as discussed by several authors,[5,6,22–25] this drying carrier improves the dryer´s performance during drying of rich sugar products; as the pineapple stem extract studied in this work. The statistical analysis performed shows that the processing parameters have significant effects on product quality. The results indicate the maltodextrin concentration of 80%, the W_S/W_max ratio of 36% and T_gi of 110°C, as the optimized conditions for production of spray-dried extract of pineapple stem. The relative enzymatic activity of the spray-dried product obtained under the optimized conditions was at least 80% of the initial activity.

ACKNOWLEDGMENTS

We gratefully acknowledge The State of São Paulo Research Foundation (FAPESP), The National Council for Scientific and Technological Development (CNPq), and The Coordination for the Improvement of Higher Education Personnel (CAPES) for financial support.