Degradation kinetics of ascorbic acid in encapsulated spray-dried honey powder packaged in aluminium laminated polyethylene and high-density polyethylene

ABSTRACT

The present study evaluated the stability of nutritionally rich encapsulated spray-dried honey powders in terms of hygroscopicity, glass transition temperature, total phenolic content, antioxidant activity and ascorbic acid using maltodextrin, gum arabic, and whey protein concentrate as carriers during a storage period of 180 days using high-density polyethylene and aluminium laminated polyethylene as packaging materials at 25°C (room temperature) and 35°C (accelerated temperature). The results revealed that temperature caused a negative influence on the glass transition temperature and stability of ascorbic acid. The kinetics of ascorbic acid degradation followed a first-order reaction with a reaction rate constant dependent on temperature and packaging material. Honey powder developed with whey protein concentrate as carrier agent and stored in aluminium laminated polyethylene pouches at 35°C possessed the highest antioxidant activity and ascorbic acid due to the presence of phenolic compounds in honey, aonla (Emblica officinalis. Gaertn), and basil (Ocimum sanctum) extract. The honey powders stored in aluminium laminated polyethylene pouches showed comparatively better antioxidant properties (total phenolic content, ascorbic acid, and antioxidant activity) and minimum hygroscopicity than the powders stored in high-density polyethylene at both the storage temperatures.

KEYWORDS:

• Honey powder
• Kinetics
• Ascorbic acid
• Antioxidant activity
• Packaging material

Introduction

Honey has been considered a very healthy food product due to the high concentration of bioactive compounds and phenolic acids.^[1] The viscous nature and tendency to crystallize makes honey cloudy, thus less appealing to the consumer and also results in higher water activity (a_w) levels which correspond to microbial contamination. The conversion of honey into a shelf-stable powder might potentially increases its storage stability thus facilitating handling, storage, transport, and export.^[2,^3] Spray drying is the most widely used technique for drying of liquid materials by atomizing the feed droplets into a drying chamber in the presence of hot air. However, the high contents of low molecular weight sugars in the honey result in highly hygroscopic and viscous products, thus posing a storage problem.^[4] Therefore, the use of high molecular weight carriers such as polysaccharides, gums and proteins becomes imperative to simplify the drying process, storage conditions and concentration of polyphenols, phenolic acids, and flavonoid compounds in the powders.^[5,^6] Basil (Ocimum basilicum) contains phenolic compounds namely eugenol, cirsilineol, rosmarinic acid, orientin and vicenin, whereas aonla (Emblica officinalis Gaertn) in addition to polyphenols, is a remarkably rich source of ascorbic acid. The stability of these bioactive compounds is further enhanced due to addition of carriers.^[2] Knowledge of degradation kinetics, including reaction order and rate constant, is essential to predict losses in essential compounds during thermal processing as well as storage under different conditions. Ascorbic acid is usually selected as an index of the nutrient quality because of its labile nature compared to other nutrients in foods.^[7] The different processing methods, packaging material and storage temperatures may lead to loss of vitamin C (ascorbic acid) due to oxidation and hydrolysis.^[8]

Packaging plays a significant role in determining the shelf stability of food powders during storage. The nature of the packaging material determines the internal environment of the package, thus determining the rate and extent of nutrient loss and microbial activity during storage.^[9] Mechanical and barrier properties of packaging material help in determining the type of package to be used for a particular food powder. A quality packaging material allows minimum water vapor permeability and restricts the water activity thus minimizing water.^[10] Several studies have been carried out to analyze the storage stability of spray-dried blueberry pomace extract, blackberry powder, yogurt powder, and tomato powder.^[6,^11–13] Spray-dried honey powder possessing antioxidant activity (AOA) and ascorbic acid content was developed with addition of aonla and basil extract in our previous study.^[14] However, no research on the determination of the storage stability of spray-dried honey powder has been carried out to date. So, the aim of the present study was to evaluate the storage stability of honey powder analyzed in terms of quality parameters (hygroscopicity, glass transition temperature [T_g], ascorbic acid, total phenolic content [TPC], and AOA) using two different packaging materials namely high-density polyethylene (HDPE) and aluminium-laminated polyethylene (ALP) pouches at 25 and 35°C for a storage period of 180 days.

Materials and methods

Helianthus annuus honey samples were collected from the local bee-keepers of Punjab, India. The botanical origin of the samples of honey was based on the pollen spectrum (45% and above), which was the ratio of the frequency of each pollen type in honey.^[15] The following terms were used for frequency classes: predominant pollen (>45% of pollen grains counted), secondary pollen (16–45%), important minor pollen (3–15%), and minor pollen (<3%).The chemicals maltodextrin (MD; DE-20), gum arabic (GA), Folin-Ciocalteau reagent, gallic acid, and sodium carbonate were procured from Loba Chemie Pvt. Ltd., Mumbai; 2,2-diphenyl picryl hydrazyl (DPPH), acetone, and methanol (high-performance liquid chromatography [HPLC] grade) were purchased from Ranbaxy, New Delhi and whey proteins concentrate (70% protein) were purchased from Mahaan Proteins Ltd., New Delhi. Aonla (Neelam variety) was purchased from Punjab Agriculture University (PAU; Ludhiana) and basil leaves (holy basil) were purchased from local farmers of Punjab, India. The packaging materials, i.e., aluminium laminated polyethylene (ALP) and HDPE were procured from Floeter India Retort Pouches Pvt. Ltd., Manesar, Gurgaon.

Sample preparation and spray drying

Based on preliminary experiments, honey was blended with water in the ratio 1:3.5 to prevent clogging of the spray dryer which would have been caused due to the highly viscous nature of honey. The three carrier agents viz. MD (DE-20; 50% w/w of honey), GA (45% w/w of honey), and whey protein concentrate (WPC; 35% w/w of honey) were subsequently added to the blend. The feed mixture was further supplemented with aonla and basil extract at 8% w/w of honey and 6% w/w of honey, respectively.^[14]

The mixtures were fed into a tall type laboratory scale spray dryer (S.M. Scientech, Calcutta, India) with concurrent arrangement and a two-fluid nozzle atomizer with nozzle tip diameter settings of 0.7 and 1.5 mm. A peristaltic pump was used for metering the feed into the dryer. Optimized conditions for production of honey powder using MD, GA, and WPC with inlet temperature, feed rate, outlet temperature, and blower speed were set at 170°C, 0.11 mL/s, 90°C and 2000 rpm, respectively. These conditions were deduced in a previous work.^[2]

Storage stability

The honey powders produced from three different carriers packed in ALP and HDPE pouches were heat sealed with minimum possible air space lingering in the leak-proof packets. Pouches prepared using each packaging material were stored in separate desiccators filled with saturated solution of MgCl₂ in order to provide relative humidity (RH) of 32.8% over a period of 180 days. Storage study was then conducted at ambient storage conditions (25°C) and accelerated storage conditions (35°C) as proposed by Labuza and Schmidt.^[16] Honey powders so produced were then periodically analyzed with respect to hygroscopicity, T_g, ascorbic acid, TPC, and AOA (at 0, 30, 60, 90, 150, and 180 days).

Hygroscopicity

One gram of sample of powder was placed at 25ºC in hermetically closed desiccators containing sodium chloride saturated solution (75% RH). After a week, the sample was weighed and hygroscopicity was expressed as grams of adsorbed moisture per 100 g dry solids (g/100 g).^[17]

T_g

Differential scanning calorimeter (Mettler Toledo DSC821, Switzerland) was used to determine the T_g of the powder samples. Four milligrams of the honey powder sample was scanned in hermetically sealed aluminium pans. An empty aluminium pan was used as a reference. The heating rate was set at 10°C/min and the samples were scanned between –10 to 120°C. The midpoint values for T_g of the samples were calculated using STARe software (Version 8.1 Mettler-Toledo, Switzerland).

TPC

Two-hundred fifty milligrams of the honey powder was mixed with 10 mL of 60% acetone and the mixture was stirred for 30 min at a temperature of 30°C. Sixty microliters of supernatant, 300 µL of Folin-Ciocalteau reagent, and 750 µL of 20% sodium carbonate in water were added to 4.75 mL of water. After 30 min, the absorbance was measured at 760 nm using an ultraviolet-visible (UV-Vis) spectrophotometer (Hach DR 6000, Germany) with methanol as the reference. Gallic acid (0–100 mg/L) was used to produce a standard calibration curve. The TPC was expressed in milligrams of gallic acid equivalents (GAEs/100 g of spray-dried powder).^[18]

AOA

Two-hundred fifty milligrams of the sample was mixed with 10 mL of 60% acetone and then the resultant mixture was stirred for 30 min at 30ºC. Two milliliters of extract was mixed with 2 mL methanolic solution containing 1 mM DPPH. The mixture was shaken dynamically and kept for 30 min. The absorbance was measured at 517 nm using a UV-Vis spectrophotometer (Hach DR 6000, Germany). The absorbance of control was obtained by replacing the sample with methanol.^[19] DPPH radical scavenging activity of the sample was calculated as follows:

Ascorbic acid

One mg honey powder was treated with 20 mL of 0.4% oxalic acid at room temperature for 5 min and thereafter filtered through Whatman No. 4 filter paper. The filtrate (1 mL) was mixed with 9 mL 2,6-dichlorophenol indophenol dye, and absorbance was recorded within 15 min at 520 nm using a UV-Vis spectrophotometer (Hach DR 6000, Germany) against a blank. Ascorbic acid was calculated on the basis of the calibration curves of ascorbic acid and was expressed as mg/100 g of ascorbic acid as the average of three replications.^[20]

Kinetics of ascorbic acid

Zero or first-order reactions represent the loss in food quality.^[21] The degradation of ascorbic acid in the encapsulated honey powders produced with different carriers was calculated by using the standard equation for a first-order kinetic model as follows:

where C is the ascorbic acid concentration at time t; C_o is the ascorbic acid concentration at time zero; k, the degradation rate constant (day⁻¹) is obtained from the slope of a plot of the natural log of C/C_o versus time; and t, being the storage time (days).The half-life corresponds to the time at which the ascorbic acid is reduced by 50% with respect to zero time and it was calculated by the equation:

The graphs of mean values were plotted and regression coefficient (R²) was calculated using Excel version 2010.

Results and discussion

Hygroscopicity

The reported values of hygroscopicity of honey powders during 180 days of storage using different packaging materials are presented in Fig. 1a and 1b. It is evident from the figures that hygroscopicity of spray-dried honey powders produced with MD, GA, and WPC as carriers, respectively, increased linearly with storage period, temperature, and type of packaging material. The hygroscopicity values of the honey powders produced with all three carriers were higher at 35°C in comparison to powders stored at 25°C. The increase in hygroscopicity of the powders during storage can be attributed to the capacity of amorphous sugars present in the honey powders to attract water molecules from the surrounding air. The lowest hygroscopicity values of honey powder were obtained with WPC as compared to other carriers due to its lower water concentration gradient. Similar results were suggested during spray drying of soya sauce by Wang et al.^[22]

Figure 1. Hygroscopicity of spray-dried honey powders produced with maltodextrin (MD), gum arabic (GA), and whey protein concentrate (WPC) carrier agents stored in different packaging materials at (a) 25°C and (b) 35°C.

It is clear from Fig. 1a and 1b that honey powders packed in ALP pouches presented lower hygroscopicity values compared to those packed in HDPP pouches at both storage temperature (i.e., 25 and 35°C).The reason behind which is lower water vapor permeability of ALP as compared to HDPE. These results are in consonance with the study of Molina et al.^[4] who reported higher hygroscopicity of dragon powder packed in polyethylene as compared to ALP.

T_g

T_g is an important parameter which controls the behavior of a food and its deterioration during processing, packaging, and storage.^[23] Fig. 2a and 2b indicate the reduction of T_g in honey powders produced with different carriers in different packaging materials regardless of the storage temperature and time. It revealed that the changes in T_g during storage were dependent on temperature and time dependent and the exposure of honey powders to higher temperatures led to decrease in flowability because of caking problem. After 120 days of storage, there was a significant reduction in T_g of honey powders which can be attributed to water absorption by hygroscopic amorphous sugars from ambient air, thus leading to formation of clusters. These results are consistent with the findings reported for spray-dried dragon fruit juice and tomato powder in which T_g decreased with storage time.^[13,^23] Honey powders produced with WPC showed the highest T_g throughout storage followed by MD and GA. This was partially due to the film formation which resulted in reduction of particle-wall stickiness and also due to higher molecular weight of WPC. Similar behavior was observed for spray-dried tamarind pulp powder and bayberry powder.^[5,^24]

Figure 2. Glass transition temperature (Tg) spray-dried honey powders produced with maltodextrin (MD), gum arabic (GA), and whey protein concentrate (WPC) carrier agents stored in different packaging materials at (a) 25°C and (b) 35°C.

The T_g-values of honey powders packed in the ALP were higher than HDPE at both storage temperatures due to lower water vapor transmission rate (WVTR) of the ALP as compared to HDPE pouches. Pua et al.^[25] also reported similar results for accelerated storage studies of jack fruit powder packed in aluminium foil laminated polyethylene pouches at 75% RH and 28°C temperature.

TPC and AOA

Figs. 3 and 4 depict the changes in TPC and AOA of nutritionally rich honey powder with respect to different carriers measured at regular intervals of 30 days over a period of 180 days of storage at 25 and 35°C in HDPE and ALP pouches. The TPC and AOA observed in the honey powders stored at 35°C was comparatively more than the powders stored at 25°C. The presence of phenolic compounds in honey, aonla extract, basil extract, and the occurrence of Maillard reaction (between sugars present in honey and protein fraction of carriers) products could have contributed to increase in the AOA. Ferrari et al.^[6] also observed that the DPPH radical scavenging activity of spray-dried blackberry powder increased as the concentration of Maillard reaction products (MRPs) increased with the increase in storage temperature, whereas the decrease of AOA of honey powders reflected the depletion of TPC and ascorbic acid developed with all three carriers after 120 days of storage at 25 and 35°C. This depletion was due to the adsorption of water which damaged the structure of the microencapsulated honey powder, thereby causing wall dissolution and oxygen diffusion into the core of microcapsule leading to oxidation. Similar results of initial increase in phenolic content and antioxidant activities and finally decreased values under prolonged storage were observed during spray drying of blueberry and pomegranate.^[26,^27] In the present study, honey powder produced with WPC as carrier agent showed the best results for the retention of phenolic content followed by MD and GA which in turn enhanced the AOA during storage (Figs. 3 and 4). Flores et al.^[28] also reported that whey protein microcapsules had higher AOA than GA microcapsules during storage of spray-dried blueberry powder. Higher retention of phenolic compounds was observed in honey powders packed in ALP pouches compared to HDPE pouches as the latter exhibited less permeability to light and oxygen against isomerization and oxidation of polyphenols. Murugesan and Orsat^[29] also reported that polyethylene was effective in maintaining the phenolic content and color of spray-dried elderberry juice powder due to its low light, humidity, and oxygen transmission rate.

Figure 3. Total phenolic content of spray-dried honey powders produced with maltodextrin (MD), gum arabic (GA), and whey protein concentrate (WPC) carrier agents stored in different packaging materials at (a) 25°C and (b) 35°C.

Figure 4. Antioxidant activity of spray-dried honey powders produced with maltodextrin (MD), gum arabic (GA), and whey protein concentrate (WPC) carrier agents stored in different packaging materials at (a) 25°C and (b) 35°C.

Ascorbic acid stability

In our previous studies, no ascorbic acid was detected in honey.^[2] Therefore, aonla extract was added to honey to produce honey powder rich in ascorbic acid which ranged from 85.26, 87.23, and 94.87 mg/100 g with MD, GA, and WPC as carrier agents, respectively.^[14]

Ascorbic acid degradation in honey powders produced with all three carrier agents exhibited good agreement with first-order kinetics (R² = 0.90–0.99; Figs. 5a, 5b, and 5c) in different packaging materials throughout storage. Different researchers reported that dried pineapple, persimmons, and kiwi fruit also followed first-order kinetics for ascorbic acid degradation.^[8,³⁰,^31]

Figure 5. Degradation kinetics of ascorbic acid of spray-dried honey powders produced with different carrier agents (a) maltodextrin (MD); (b) gum arabic (GA); (c) whey protein concentrate (WPC) stored in different packaging materials at 25°C and 35°C.

The kinetics of degradation of ascorbic acid was monitored over the storage period, rate constants and half-life values of reactions in spray-dried honey powders (Table 1). During storage at 25 and 35°C, honey powder produced with WPC packed in ALP had the longest half-life, followed by those produced with GA and MD as carrier agents. However, honey powders produced with MD as carrier agent and packed in HDPE resulted in a higher reaction rate constant. The higher half-life of honey powder produced with WPC as carrier agent, in both packaging materials was attributed to its good emulsifying capacity and protein–polysaccharide matrix. Similarly, Kha et al.^[32] also reported that improved resistance of carotenoids in encapsulated gac oil powder to oxidation could be achieved with encapsulation. The lowest half-life value of spray-dried honey powders was observed in the powders developed with MD. This can be attributed to the degradation of ascorbic acid due to hydrolytic reaction and increase in the moisture content of dried product, which favored oxygen diffusion toward the encapsulated material, thus increasing in the rate of physicochemical changes, such as collapse, caking, agglomeration, browning, and oxidation.^[34] The degradation of honey powders stored at 35°C was higher than those stored at 25°C due to high molecular mobility and accelerated oxidation reactions which led to degradation of ascorbic acid. Some studies reported a logarithmic course of ascorbic acid destruction with an arithmetic increase in temperature.^[33,^34]

Table 1. Kinetic parameters for ascorbic acid degradation in spray-dried honey powders produced with maltodextrin (MD), gum arabic (GA), and whey protein concentrate (WPC) carrier agents stored in different packaging materials at 25 and 35°C.

Sample	T°C	Packaging material	Rate constant (k) (days^−1)	Half lives(t1/2) (days)	Regression coefficient R^2
MD	25	HDPE	0.0045	161.45	0.97
	25	ALP	0.0037	196.3	0.97
	35	HDPE	0.0059	120.45	0.95
	35	ALP	0.0053	136.47	0.95
GA	25	HDPE	0.0039	181.8	0.98
	25	ALP	0.0032	216.38	0.98
	35	HDPE	0.0063	112.75	0.99
	35	ALP	0.0036	193.34	0.99
WPC	25	HDPE	0.0031	224.86	0.97
	25	ALP	0.0023	310.71	0.99
	35	HDPE	0.0035	197.11	0.96
	35	ALP	0.0025	280.79	0.97

Honey powder stored in ALP pouches showed lesser degradation of ascorbic acid compared to that stored in HDPE pouches at 25 and 35°C (Figs. 5a, 5b, and 5c). This trend resulted due to the lower oxygen transmission rate of ALP pouches compared to HDPE pouches hence, retarding oxidation of ascorbic acid. Kha et al.^[35] also reported enhanced loss of total carotenoid content and AOA of spray-dried gac powder non-laminated bags as compared to laminated aluminium bags under similar storage conditions.

Conclusion

It could be concluded that there was a progressive degradation of ascorbic acid content, which was best represented by first-order reaction in honey powders, due to the increase in the storage temperature and period. Honey powder produced with WPC as carrier showed the longest half-life lowest ascorbic acid degradation rate at 25°C and addition to the highest T_g among other carriers. The results also established that the ALP due to its lower oxygen transmission rate and water vapor permeability is recommended for long-term storage of honey powders among the two packages used. The development of spray-dried honey powders using all three carrier agents and with the addition of aonla and basil extract were found to be rich in ascorbic acid and AOA up to 120 days of storage, hence of better utility as ingredients to different food products.